WO2011104565A1 - Anticorps de camélidés destinés à être utilisés dans des compositions et procédés pour le traitement du cancer - Google Patents

Anticorps de camélidés destinés à être utilisés dans des compositions et procédés pour le traitement du cancer Download PDF

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WO2011104565A1
WO2011104565A1 PCT/GB2011/050382 GB2011050382W WO2011104565A1 WO 2011104565 A1 WO2011104565 A1 WO 2011104565A1 GB 2011050382 W GB2011050382 W GB 2011050382W WO 2011104565 A1 WO2011104565 A1 WO 2011104565A1
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cells
camelid
cancer
specific
taa
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Sabah Abdel Amir Jassim
Zeyad Ahmed Shabeeb Al Juboori
Ahmed Sahib Abdulamir
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Arab Biotechnology Company
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6821—Plant heterodimeric toxins, e.g. abrin or modeccin
    • A61K47/6823—Double chain ricin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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
    • A61K47/6853—Carcino-embryonic antigens
    • 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
    • A61K47/6867—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 a cell of a blood cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary

Definitions

  • the invention relates to the prevention and treatment of disease. More specifically, the invention relates to novel compositions and methods for immunotherapy, in particular, cancer immunotherapy.
  • Cancer is a group of diseases in which cells are aggressive (grow and divide without respect to normal limits), invasive (invade and destroy adjacent tissues), and sometimes metastatic (spread to other locations in the body). These three malignant properties of cancers differentiate them from benign tumours, which are self-limited in their growth and don't invade or metastasize (although some benign tumour types are capable of becoming malignant). Cancer may affect people at all ages, even foetuses, but risk for the more common varieties tends to increase with age. Cancer causes about 13% of all death. According to the American Cancer Society, 7 million people died from cancer in the world during 2007. Apart from humans, forms of cancer may affect other animals and plants (Valsecchi and Steliarova-Foucher, 2008).
  • cancers are caused by abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens, such as tobacco smoke, radiation, chemicals, or infectious agents. Other cancer-promoting genetic abnormalities may be randomly acquired through errors in DNA replication, or are inherited, and thus present in all cells from birth. Complex interactions between carcinogens and the host genome may explain why only some develop cancer after exposure to a known carcinogen. New aspects of the genetics of cancer pathogenesis, such as DNA methylation, and micro RNAs are increasingly being recognized as important (Croce, 2008).
  • Cancer-promoting oncogenes are often activated in cancer cells, giving those cells new properties, such as hyperactive growth and division, protection against programmed cell death, loss of respect for normal tissue boundaries, and the ability to become established in diverse tissue environments.
  • Tumour suppressor genes are often inactivated in cancer cells, resulting in the loss of normal functions in those cells, such as accurate DNA replication, control over the cell cycle, orientation and adhesion within tissues, and interaction with protective cells of the immune system (Ree et al., 2008).
  • Cancer is a major health concern worldwide. To date, the morbidity and mortality associated with cancer is second in frequency only to heart disease. With increases in effective management of heart disease and projected increases in average life expectancy, cancer looms as the number one health concern for the near and distant future.
  • Antibodies also known as immunoglobulins, abbreviated Ig
  • Ig immunoglobulins
  • Ig immunoglobulins
  • They are typically composed of two identical large 'heavy' (H) chains and two identical small 'light' (L) chains, joined by disulphide bonds to form a classic Y shaped molecule.
  • H and L variable (V) regions on the H and L chains
  • VH and VL variable regions on the H and L chains
  • C- terminal constant (C) region of the polypeptide chains demonstrates little variability between immunoglobulins of the same class or type.
  • Immunoglobulin molecules are classified according to the type of H chain: immunoglobulin G (IgG), IgA, IgM, IgD and IgE.
  • the IgG molecules are further categorized into four subclasses according to amino acid sequence. The most conspicuous differences between lgG1-4 lie in the amino acid composition of the 'hinge region' between the first and second constant domains. Structural differences between the IgG subclasses are also reflected in their susceptibility to proteolytic enzymes.
  • Antibodies are a key component of the adaptive immune response, playing a central role in both in the recognition of foreign antigens and the stimulation of an immune response to them. It is not surprising therefore, that many immunotherapeutic approaches involve the use of antibodies. The advent of monoclonal antibody technology has made it possible to raise antibodies against specific antigens such as the unusual antigens that are presented on the surfaces of tumours.
  • Immunotherapy is the use of the immune system to reject the malicious cells, say in cancer, or microbial pathogenic antigens like in infectious diseases.
  • the main premise is stimulating the patient's immune system to attack the target e.g. malignant tumour cells that are responsible for the disease. This can be either through immunization of the patient, in which case the patient's own immune system is trained to recognize tumour cells as targets to be destroyed, or through the administration of therapeutic antibodies as drugs, in which case the patient's immune system is recruited to destroy tumour cells by the therapeutic antibodies.
  • Radio-immunotherapy involves the use of radioactively conjugated murine antibodies against cellular antigens. Most research currently involves their application to lymphomas, as these are highly radiosensitive malignancies. To limit radiation exposure, murine antibodies were especially chosen, as their high immunogenicity promotes rapid clearance from the body.
  • tumour cells Since the immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self, many kinds of tumour cells that arise as a result of the onset of cancer are more or less tolerated by the patient's own immune system since the tumour cells are essentially the patient's own cells that are growing, dividing and spreading without proper regulatory control.
  • tumour cells display unusual antigens that are either inappropriate for the cell type and/or its environment, or are only normally present during the organisms' development (e.g. foetal antigens).
  • antigens include the glycosphingolipid GD2, a disialoganglioside that is normally only expressed at a significant level on the outer surface membranes of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier.
  • GD2 is expressed on the surfaces of a wide range of tumour cells including neuroblastoma, medulloblastomas, astrocytomas, melanomas, small-cell lung cancer, osteosarcomas and other soft tissue sarcomas. GD2 is thus a convenient tumour-specific target for immunotherapies.
  • tumour cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signal transduction pathways that cause the unregulated growth and division of the tumour cell.
  • Examples include ErbB2, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of breast cancer tumour cells.
  • T cells recognize antigen as small peptides bound to cell surface molecules encoded by the major histocompatibility gene complex (MHC).
  • MHC major histocompatibility gene complex
  • TCR T-cell receptors for antigen (TCR) of cytotoxic T lymohocyte (CTL) bind to a molecular complex on the surface of the antigen-presenting cells (APC) formed by peptide epitopes usually derived from viral or TAA and MHC class I molecules.
  • APC antigen-presenting cells
  • the peptides that are recognized by CTL are usually fragments 8- 10 residues long that associate non-covalently with polymorphic class I MHC molecules (Rammensee et al., 1993).
  • helper T lymphocyte(HTL) express the CD4 surface marker and recognize slightly larger peptides (12-20 residues) in the context of HC class II molecules, which are only expressed in specific types APC such as B-lymphocytes, monocytes/macrophages and DC (Germain and Margulies, 1993; Shebzukhov et al., 2007).
  • MHC binding peptides which are transported to the APC surface for presentation to the TCR (Germain and Margulies, 1993; Rammensee et al., 1993; Shebzukhov et al., 2007). After TCR engagement by appropriate MHC-peptide complexes, CTL have the ability to bind and kill target cells expressing foreign (infectious) or TAA.
  • HTL MHC-peptide recognition by HTL
  • these cells produce lymphokines that enhance and amplify CTL immune responses, or in some cases, HTL may also induce the lysis of the cell presenting the antigen or bystander cells via cytolytic mechanisms such as TNF and Fas ligand (Malyankar, 2007; Rammensee et al., 1993).
  • TAA Some of the changes that occur during cell transformation can produce MHC-binding peptides that are immunogenic for CTL or HTL.
  • TAA include: (1 ) oncogenic viral proteins; (2) abnormal over-expression of foetal or tissue specific proteins; and (3) mutated or over-expressed oncogene or tumour suppressor gene products (Boon et al, 1994; Fisk et al., 1995; Urban and Schreiber, 1992).
  • TAA such as CEA, PSA, HER2 and p53, which serve as "tumour markers” have been identified and biochemically characterized (Cheever et al., 1993; Nijman et al., 1994; Yoshino et al., 1994).
  • the invention arises from the inventors' finding that, by immunization of an adult camel with synthetic carcinoembryonic antigen (CEA) or a tumour associated antigen purified and eluted chemically from human cancerous cells, it is possible to produce serum containing high level of anti-CEA or anti- TAA IgG antibodies.
  • Anti-TAA antibodies were also present in the milk and were passed from mother to calf via udder miik feeding. High levels of antibodies were also detected in the urine of the immunized camel.
  • the invention provides a method for producing immunoglobulin that specifically binds to cancer cells, said method comprising immunising a camelid with tumour associated antigen purified from cancer cells by acid elution and purifying tumour associated antigen-specific immunoglobulin from a body fluid of said camelid.
  • Cancerous cells from which TAAs may be isolated by acid elution include, for example, acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) cells.
  • AML acute myeloid leukaemia
  • CLL chronic lymphocytic leukaemia
  • Camelids are members of the biological family Camilidae, which includes Camelus dromedarius (the dromedary or Arabian camel, with one hump), Camelus bactrianus (the Bactrian camel, with two humps), Lama pacos (the alpaca), Lama guanicoe (the guanaco), Lama peruana (the llama) and Vicugna vicugna (the vicuna). Camelids are even-toed ungulates classified in the order Artiodactyla.
  • VHH VH domain of a conventional antibody and that of a heavy-chain antibody of Camelidae the latter is referred to herein as VHH.
  • the heavy-chain antibodies from Camelidae behave quite differently in comparison with conventional four-chain antibodies. More specifically, the heavy-chain antibodies acquired the potential to recognize protein cavities and as such the ability to inhibit enzymes (Hamers-Casterman et al., 1993; Jassim and Najii 2001). The structure of a dromedary VHH in complex with lysozyme revealed the unusual convex surface topography of the antigen- combining site of this single-domain antibody fragment (Desmyter et al., 1996).
  • camelid immune system is very effective in producing highly stable, specific and low molecular weight IgG antibodies against acid eluted tumour associated antigen.
  • the study described herein demonstrates that camelids can be used as a potent source for the production of specific IgG antibodies against cancer cells.
  • TAA-specific camelid IgGs described herein may be applied to any kind of human or animal cancer cells.
  • Cancers which may be treated by the method of the invention include, but are not limited to, ovarian cancer, bladder cancer, lung cancer, cervical cancer, breast cancer, prostate cancer, glioma, fibrosarcoma, retinoblastoma, melanomas, soft tissue sarcomas, osteosarcoma, colon cancer, carcinoma of the kidney, pancreatic cancer and leukaemia (e.g. AML and CLL).
  • Camelids are particularly advantageous for the production of unique antibodies (lgG2 immunoglobulin) specific towards TAA or tumour specific antigens (TSA) that can recognise TAA peptides complexed with MHC molecules without the need for reacting with MHC.
  • TAA tumour specific antigens
  • This invention provides the scientific and clinical background for global treatment of different types of human malignancies without further immunological preparation of each patient as might be needed when human antibodies are used in immunotherapy of cancers because camelid IgG's convex, small and efficient antigen biding site can bind and react to the specific epitopes without association with MHC class I and II haplotype restriction. Therefore, there will be global camelid anticancer IgGs for each cancer type.
  • tumour associated antigen used for specific camelid IgG production is a tumour associated peptide extracted from cancer cells, e.g. from tumour tissue. Such peptides are preferred to synthesized tumour peptides which may lack the natural conformational configuration which acts in concert with the amino acid sequence for epitope designation and recognition by the paratope of the antigen binding site of the binding antibody.
  • the TAA is extracted from tumour tissue or cancerous cells by acid elution.
  • the antigen is coupled to a carrier protein for camelid immunisation: the inventors have found EDC-protein coupling TAA-carrier protein with Freunds to be effective.
  • antigen e.g. TAA
  • TAA camelid autologous dendritic cells are used to stimulate the other arm of the immune system. Most preferably, these two methods of immunisation are combined to yield robust antigen-specific antibody production in the immunised camelids.
  • This methodology for producing highly specific and stable camelid IgG against TAA may be applied to different kinds of epitopes such as surface receptors expressed excessively on tumour cells, as antagonist for receptors like EGFRII, Tyrosine kinase receptors etc. or as agonist for apoptosis receptors like FAS (CD95) and TNFRII, etc., which are able, upon stimulation, to induce apoptosis via the exogenous pathway of apoptosis (FRAD- and TRAD- associated domains).
  • epitopes such as surface receptors expressed excessively on tumour cells, as antagonist for receptors like EGFRII, Tyrosine kinase receptors etc. or as agonist for apoptosis receptors like FAS (CD95) and TNFRII, etc.
  • TAA elution method is used to elute the surface antigens bound to MHC class I or II or freely expressed on the cell membrane
  • this invention can also be used for locating the HIV surface glycoproteins like gp120 expressed on the surfaces of CD4+ and macrophages cells of HIV infected patients.
  • the method of this invention can be used to trace, bind and stimulate (agonist effect) various surface receptors (antigens) in attempt to treat several metabolic and endocrine disease like targeting insulin receptors in diabetes mellitus or as antagonist effect to block unwanted receptors like in autoimmune (idiopathic) cardiomyopathy.
  • This invention can be used to exploit the convex well-fitting small antigen biding site of camelid IgGs to bind and block unwanted circulating human antibodies like autoreactive antibodies in systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • This task proved to be unsuccessful by using human blocking antibodies because of the same species weak reaction and because of loose binding due to the loss of the key and whole conformity of 2 concave antigenic binding sites.
  • the invention further provides a cancer cell-specific immunoglobulin produced by the method of the invention.
  • TAA-specific camelid IgGs can be labelled with cytotoxic agents (to produce an immunotoxin), which would then be specifically delivered to the tumour cells, or with highly immunogenic molecules in attempt to convert cancerous cells to highly visible targets in front of the immune system surveillance mechanisms.
  • Cytotoxic agents with which the TAA-specific camelid IgGs could be labelled include, but are not limited to CD8+ CTLs, patient-autologous natural killer cells and apoptosis ligands (FASL), TNF, etc.
  • cytokines are needed. While the therapeutic administration of such cytokines may cause systemic inflammation, resulting in serious side effects and toxicity, a new generation of chimeric molecules can be used in the invention consisting of an immune-stimulatory cytokine attached to an antibody that targets the cytokine's activity to a specific environment such as a tumour, thus generating a very effective yet localized immune response against the tumour tissue, destroying the cancer-causing cells without the unwanted side-effects.
  • the targeted delivery of cytokines by anti-tumour antibodies is one example of using antibodies to deliver payloads rather than simply relying on the antibody to trigger an immune response against the target cell.
  • the antigen-specific immunoglobulin may be conjugated with a chemotaxic or radiotoxic moiety.
  • a preferred cytotoxic moiety is ricin A-chain.
  • the invention also provides an immunotoxin comprising a cancer cell-specific immunoglobulin produced by the method of the invention, which is conjugated to a cytotoxic moiety e.g. ricin A-chain.
  • a cytotoxic moiety e.g. ricin A-chain.
  • the anti-TAA camelid IgG antibodies labelled with a chemotherapeutic or radio-therapeutic molecule may be engineered in such a way that the antibody-payload pair separates after entry into a cell by endocytosis to increase the efficacy of the payload.
  • One strategy to accomplish this is the use of a disulfide linkage which could be severed by the reducing conditions in the cellular interior.
  • Camelid IgG against acid eluted TAA can be developed for rapid diagnostic and recognition of cancerous cells more accurately and specifically.
  • the TAA- specific camelid IgGs may be used in diagnosing the tiny foci of primary or secondary metastasis tumors anywhere in the body. This is achieved by labeling the TAA-specific camelid IgGs with radio-opaque or radioactive material that can be detected easily by X-ray or/and radioactive detection devices. This can be termed as "in vivo immunodiagnostic imaging technique".
  • the cancer cell-specific camelid immunoglobulins may be broken down (e.g. chemically or enzymatically) into small domain IgG.
  • the therapeutic or selectivity index of the isolated camelid immunoglobulins, or the labelled camelid immunoglobulins or small domains - i.e. cytotoxicity to tumour cells/cytotoxicity to normal cells - may be determined by routine assays.
  • the therapeutic of selectivity index of the camelid immunoglobulins of the invention is expected to be high in comparison to antibodies known in the art.
  • the invention provides a method for treating or preventing cancer in an individual which comprises administering to said individual an effective amount of a cancer cell-binding camelid immunoglobulin as described herein, which may optionally be conjugated to a cytotoxic moiety to form an effective immunotoxin.
  • the inventors have found that, in the antigen-immunized camel, very high levels of antigen-specific IgG antibodies were present in the serum, the milk and the urine so that antigen-specific antibodies could be readily purified from any of these fluids, which are easily collected from the animal.
  • the inventor's novel finding of significant quantities of antibody in the urine opens up a convenient opportunity for collecting antibody- containing fluid for extraction of antibodies since one adult camel excretes up to 8-25 litres of urine per day.
  • the inventors found that a camel excreting a large quantity of antibodies in the urine does not show any depletion of antibody in the serum or milk, indicating that the camel can act as a very productive biological system for producing antigen-specific antibodies.
  • the invention further provides a nutritional supplement or functional food for the treatment or prevention of a target cancer or pathogen infection, said nutritional supplement or functional food comprising one or more target-specific immunoglobulins isolated from a camelid that has been immunised with one or more antigens associated with said cancer or expressed by said pathogen.
  • the nutritional supplement or functional food comprises camelid's milk or a purification product thereof.
  • the nutritional supplement or functional food may comprise target antigen-specific immunoglobulins isolated from camelid urine.
  • the invention provides a B cell specific for tumour associated antigen, which has been isolated from a camelid which has been immunised with said tumour associated antigen. DNA extraction from said isolated B cells is envisaged for the purpose of cloning TAA-specific immunoglobulin genes.
  • the invention provides a method for producing a library of tumour associated antigen-specific immunoglobulins, which comprises the steps of:
  • tumour associated antigen-specific immunoglobulin genes into competent E. coli cells to create a gene library of tumour associated antigen-specific camelid immunoglobulins.
  • This method may be used to formulate a complete set of camelid IgGs and in particular the subclass lgG2 monoclonal antibodies against each type of human cancer which are able to destroy tumor cells by the easy recognition by the camelid IgGs in a way leading to complete destruction of the tumor primary or even the secondary metastasized foci.
  • Mass-production of TAA-specific camelid IgGs without the need to immunize camel animals again may be achieved by formulating TAA-specific clones of transformed E. coti cells or bacteriophages that produce in large quantities bioengineered anti-TAA camelid IgGs.
  • Figure 1 shows statistical analysis of CEA immunization of adult camel and passive immunization to camel calf.
  • the histogram shows the comparative differences in mean ⁇ STEM of immunized camel serum (ICS), immunized camel mi!k (ICM), immunized camel urine (ICU), immunized camel calf serum (ICCS), pre-immunized camel serum (PCS), pre-immunized camel calf serum (PCCS), and negative control blocking buffer (BB).
  • the assigned P values are the only significant differences in this figure.
  • Figure 2 shows ELISA OD values of anti-TAA specific IgG antibodies detected in serum, milk, and urine fluids of already immunized camel animals with acid eluted TAA of both CLL (black bars) and AML (grey bars) malignancies of human, as well as negative and positive controls.
  • Figure 3 shows concentration of anti-TAA 1 gG2 antibodies (ng/ml) from serum, milk, and urine fluids of already immunized camel animals with acid eluted TAA of both CLL (black bars) and AML (grey bars) malignancies of human, as well as negative and positive controls.
  • the objective is to make a tumor type specific set of anti-TAAs camelid IgG antibodies to augment the chances of tumor cell recognition.
  • the aim is to produce in the region of 2-5 monoclonal camelid IgG for each tumor type; each one specific against certain TAA of that tumor type and completely negative against eluted surface antigens of the normal cells of the same affected organ and tissues in order to achieve full cancer- oriented and specific camelid IgGs.
  • the invention relied on two cancer models; the first involved using the CEA antigen (Sigma-Aldrich) as a cancer model oncogen through injecting to the newly delivered camels in the proper scheduled immunization protocol; the second, involved using chemically eluted TAA (acid elution method) from cancerous cells of AML and CLL.
  • CEA antigen Sigma-Aldrich
  • TAA acid elution method
  • Carcinoembryonic antigen is highly expressed on cancer cells of epithelial origin, such as colorectal, lung, breast, and ovarian carcinoma (Hammarstrom 1999, Cortez-Retamozo et al 2004). It is not expressed in other cells of the body except for low-level expression in gastrointestinal tissue. This expression profile makes it an attractive target for tumour therapy.
  • AML Acute myeloid leukaemia
  • CLL chronic lymphocytic leukaemia
  • the procedure was done under sterile conditions by using ultraviolet hood (Dalton USA) and was performed as following: fresh heparinized blood was poured into a 15 ml tube and an equal volume of RPMI 1640 (Sigma- Aldrich, USA) was added and mixed well. The diluted blood was added slowly by Pasteur pipette to an equal volume of a ficoll hypaque (Pharmacia, Sweden) layer in a tube to create a top layer of diluted blood that interfaces with the below layer of ficoll. Layered tubes centrifuged with ficoll hypaque at 700 x g for 30 minutes at 4-5°C.
  • Complete RPMI 1640 media was consisted of : RPMI 1640 (Flow laboratories, UK), L-Glutamine (BDH, UK), Sodium bicarbonate (NaHC03) of final concentration 2 g/Liter (Sigma, USA), Hepes buffer 10 mM (Flow laboratories, UK), 10 % foetal calf serum (Sigma, USA), Penicillin G solution (final concentration 0.1 mg/ml), and Streptomycin solution (final concentration 0.1 mg/ml). Finally the lymphocytes suspension was adjusted using haemocytometer chamber (Fischer, USA) at a titer of 1-2 x 106 cells/ml (Kutvirt et al., 1993).
  • Leukopheresis was accomplished using the Centrifuge (American Instrument Company, SilverSpring, Md, USA). Briefly the method of Fay et al (1979) was used, as follows: blood was removed via the antecubital veins, anticoagulated with heparin and acid-citrate dextrose, pumped at 40-60 ml/min through the centrifuge bowl spinning at 500-600 x g, and returned to the patient's opposite arm. Leukocytes were removed from the buffy-coat interface by a peristaltic pump into 600-ml Fenwal bags. Seven to ten litres of blood were processed during each leukopheresis. Complete blood counts and white cell differentials were performed on the patient before and after each procedure.
  • the tumor cells were washed three times with NaCL 0.9% and the tumor-associated peptides were isolated using acid tumor peptide elution. Aliquots of 5 x 109 tumor cells were pelleted, frozen, and stored at - 80°C. Pellets were thawed and resuspended in 10 ml citrate-phosphate buffer (0.131 mol/L citric acid/0.066 mol/L Na2HP04, pH 3.3) containing protease inhibitors and were immediately centrifuged at 15,000 x g for 15 min at 4°C. Peptides were eluted from these columns using 3 ml 60% acetonitrile (Sigma, USA) in water.
  • the elute was collected in five vials (Minisorp; Nunc, Roskilde, Denmark), each containing peptides extracted from approximately 10 9 leukaemic [acute myeloid leukaemia (AML)] or lymphocytic [chronic lymphocytic leukaemia (CLL)] blasts, and then part of elutes were lyophilized (Storkus et al., 1993). TAA Lyophilization
  • TAA peptide acid elution The proteins of TAA obtained by TAA peptide acid elution were subjected to freeze drying in order to get TAA powder which is more suitable for formulating any desired concentration of TAA proteins solution for the coming steps.
  • TAA powder which is more suitable for formulating any desired concentration of TAA proteins solution for the coming steps.
  • frizzed cubes were placed in the lypholizer beakers and the latter were placed in lypholizer (Fischer, USA) and ran with the sucking power to create a negative pressure and reduce temperature to -70°C for 36 hrs.
  • the designed immunization is novel and engineered to be highly competent in producing a broad range of specific anti-TAA camelid IgG antibodies that can recognize a number of TAAs or TSAs. This is vital to override the most disappointing obstacles in immunotherapy which is the escape loss variants of cancer in that cancer vaccines target just one tumor antigen are likely to be less effective. Tumors are highly heterogeneous and antigen expression differs markedly between tumors (even within deposits in the same patient). The most effective immunotherapeutic approach is likely to raise an immune response against a broad range of tumor antigens to minimize the chance of the tumor being able to mutate and become resistant to the therapy. Hence, the intended immunization of the camel animal is designed to deploy whatever possible of the creative immunization strategies. Therefore it is intended to use the carrier protein-TAA immunogen with complete Freunds' adjuvant, This line of immunization is necessary for triggering the camel immune system in reacting with the introduced foreign human TAAs.
  • TAA are peptides or small proteins that carry the epitopes exposed on the outer surface of the cancer cells in complex with MHC I or II. Therefore they are considered as haptens which are equal to 10 A.A or ⁇ 5,000 Dalton. Hapten cannot elicit an antibody response without being attached to a carrier protein.
  • An immunogen is created by conjugating a hapten to a carrier protein. Small molecules such as drugs, organic compounds, and peptides and oligosaccharides with a molecular weight of less than 2-5 kDa are not usually immunogenic, even when administered in the presence of adjuvant. In order to generate an immune response to these compounds, it is necessary to attach them to a protein or other compound, termed as a carrier that is immunogenic.
  • the animal may also be used to create a multivalent antigen that will be able to form large antigen-antibody complexes.
  • carrier proteins the animal forms antibodies to the carrier protein as well as to the attached hapten. It is therefore important to select a carrier protein for immunization that is unrelated to proteins that may be found in the assay sample. If haptens are being conjugated for both immunization and assay, the two carrier proteins should be as different as possible. This allows the antiserum to be used without having to isolate the anti-hapten antibodies from the anti-carrier antibodies.
  • the procedure of conjugation of TAA with carrier proteins to produce immunogen included complete Freunds' adjuvant.
  • Keyhole Limpet Hemocyanin (KLH) was used in the immunization of camel animals.
  • KLH is a complex, high-MW protein widely used as a carrier protein in antibody production because of its excellent immunogenicity conferred to the attached antigens.
  • Bovine Serum Albumin (BSA) is a convenient, highly soluble protein for use as a carrier in preparing immunogens for antibody production from haptens and other non-immunogenic antigens.
  • BSA was used as non-relevant carrier proteins to assess anti-peptide antibody titers.
  • BSA was used as blocking protein in this procedure.
  • a vial of Maleimide Activated KLH (Maleimide Activated BSA, KLH conjugation Kit, Sigma, USA) was slowly opened to release the vacuum.
  • the contents of the viai was reconstituted with 1 ml of deionised water to obtain a 5 mg/ml solution of maleimide activated KLH in 20 mM sodium phosphate buffer (Sigma, USA), 230 mM NaCI, 2 mM EDTA (Sigma, USA) and 80 mM sucrose (Sigma, USA), pH 6.6. They were mixed gently without vortex and were used immediately.
  • the conjugation buffer was reconstituted in 10 ml of deionised water to obtain a solution of 20 mM sodium phosphate buffer, 100 mM EDTA and 80 mM sucrose, pH 6.6. 4 mg of TAA were dissolved in 0.5 ml of conjugation Buffer or alternatively in deionised water.
  • Peptide solution was immediately mixed with the Maleimide Activated KLH solution in a reaction vial equipped with stirring bar. Sample was De-gassed, while stirring, under a gentle nitrogen stream for 1-2 minutes. Reaction vial was caped and continued stirring for 2 hours at room temperature or overnight at 2-8°C.
  • Protein Assay (Biuret Method)
  • a formula for biuret reagent is (per litre final volume) 9 gm Sodium potassium tartrate (BDH, UK), 3 gm Copper sulfate (BDH, UK), 5 gm Potassium iodide (BDH, UK), all dissolved in 400 ml 0.2 M NaOH (BDH, UK) before bringing to final volume.
  • the volume can be scaled up or scaled down as need, Layne (1957).
  • the volumes of sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
  • the spectrophotometer was Warm up for 15 min before use.
  • the standards from bovine serum albumin were prepared and preferably calibrated using absorbance at 280 nm and the extinction coefficient.
  • 5 ml colour reagent was used to 1 ml sample as the recommended range was 0.5 to 20 mg protein.
  • a reference tube was prepared with 1 ml buffer. Unknowns were diluted to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated. Then 1 ml sample was used per assay tube.
  • Nine millilitre Biuret reagent was added to each tube, vortex immediately, and was let stand 20 min then the mixture was read at 550 nm.
  • camel (Camelus dromedarius) was injected with 10 mg to 50 mg of the formed immunogen (CEA and adjuvant) at days 0, 7, 14, 21 , and 28.
  • camels were injected with the formed immunogen (TAA, carrier protein KLH and adjuvant) at days 0, 7, 14, and 21 . This period can result in successful immunization.
  • Each camel was injected with one type of TAA solution (AML or CLL) mixed from the individual TAA solutions of five patients.
  • TAA solution AML or CLL
  • camel was injected with CEA antigen and complete Fruend's adjuvant at first injection and completed with CEA antigen and incomplete Fruend's adjuvant for the subsequent injections.
  • IgG subclasses were purified from the dromedary serum, milk and urine by differential absorption on Protein A and Protein G.
  • the lgG1 subclass contains the conventional heterotetrameric antibodies composed of two light and two heavy chains, whereas lgG2a, lgG2b and lgG3 are the homodimeric heavy-chain antibodies, devoid of light chains.
  • the IgG subclasses were obtained by successive affinity chromatography on 1 ml HiTrap Protein G and Protein A columns (Sigma- Aldrich, USA). lgG3 and lgG1 were eluted from the Protein G column with an acetate buffer (pH 3.5) and a glycine- HCI buffer (pH 2.7), respectively. The flow-through was loaded on the Protein A column to recover two more fractions of heavy-chain antibodies, lgG2a and lgG2b which were recovered with the acetate buffer at pH 4.5 and 3.5, respectively. The IgG protein concentrations were determined spectrophotometrically, assuming an ⁇ % of 13.5 at 278 nm for all subclasses.
  • the conventional lgG1 antibodies were expected in the range of M T 160 000 Da, huge amounts of heavy-chain antibodies with M T of -95 000 Da, whereas, lgG2 and lgG3 of dromedary were the monomeric heavy chains of ( r 45 000 and 42 000 Da, respectively). Therefore, the protein band at molecular weight of 45,000 and 42,000 Da was eluted as shown earlier for further ELISA validation of these lgG2 and lgG3 against TAA.
  • Indirect ELISA was used to measure the anti- CEA antigen as follows: Three microtiter plates were coated with the CEA antigen. The studied samples and controls were carried-out in duplicate. CEA was adsorbed onto a 96-microtiter plate in carbonate buffer (pH 9.6) overnight at 4°C. Bovine serum albumin (BSA; 67,000 M.W.) is often used as non-relevant protein to assess anti-CEA antibody titers. BSA was used as blocking buffer, 10 mg/ml in PBS/Tween-20 and 50 ⁇ were added for 1 h at 37"C. Non-specific binding sites on the microtiter plate were blocked by using a blocking buffer after coating the CEA antigen to the plate.
  • BSA bovine serum albumin
  • CEA-coated microtiter plates After washing step, different animal fluid specimens, withdrawn from the immunized camels and its baby were added onto the CEA-coated microtiter plates. These samples included serum, milk and urine of immunized camel and the serum of a camel baby. Immunized serum, milk, urine, and baby's serum were diluted 1 :200 respectively, and 100 ⁇ of each dilution were added on the coated microtiter plate to bind the adsorbed TAA for 2 hours at 37°C. Any CEA non-specific antibodies or unbound were washed away by washing step three times with phosphate buffer saline (PBS; Fluka-Switzerland) containing 0.05% Tween-20 (BDH-UK) before the next assay step.
  • PBS phosphate buffer saline
  • BDH-UK Tween-20
  • the negative control was divided into three categories to exclude all the misdiagnosis and cross reaction possibilities or the false positive and ⁇ or negative possibilities.
  • Category (A) included serum from pre-immunized camel and category (B) included serum from pre- immunization baby.
  • category (C) included blocking buffer instead of serum.
  • 100 ⁇ of secondary antibody-enzyme conjugate (goat anti-human IgG (y- chain specific peroxidase conjugate, Sigma- USA) in 1:30 000 PBS Tween-20 was used to bind the anti-CEA antibody for 1 hour at 37°C. After washing, the bound antibodies were detected by adding TMB substrate. Then the reaction was measured by using ELISA reader (Bioteck, UK) at wavelength 450 nm.
  • One immunization protocol may produce an antibody titer/ the higher the dilution factors, the stronger the polyclonal immune response (Lateef et al., 2007). Measurement of the Anti-TAA Ab Response Via Camel's Fluids and Fractionated lgG2 Antibodies
  • Antibodies produced against immunogen bind to both peptide (hapten or epitope) and to carrier protein.
  • the coated TAA peptides on ELISA solid phase were coupled with non-relevant carrier protein BSA using the same coupling chemistry (maleimide) of the injected immunogen. The procedure
  • TAA was adsorbed onto a 96-microwell plate in carbonate buffer (pH 9.6) overnight at 4°C.
  • Fifty microlitre of BSA and 10 mg/ml in PBS Tween20 were added at 37°C for 1 h which was used as blocking buffer.
  • PBS containing 0.05% Tween20 different specimens of immunized camel fluids or the fractionated and purified lgG2 antibodies were added onto the TAA-coated microtiter plates.
  • the fractionated lgG2 antibodies were diluted 1 : 200 from the original concentration, 250 ng/ml, and 100 ⁇ of this dilution were added.
  • Any TAA non-specific antibodies like anti- mcKLH rather than anti-TAA antibodies were washed away by washing step three times with PBS containing 0.05% Tween-20 before the next assay step.
  • the negative control was divided into three categories to exclude all the misdiagnosis and cross reaction possibilities or the false positive and ⁇ or negative possibilities.
  • Category (A) included the acid eluted MHC class I and ll-attached peptides from mixture of normal leukocytes and lymphocytes from normal subjects.
  • Category (B) included serum from pre-immunization camel.
  • category (C) included blocking buffer instead of serum.
  • As a positive control some wells of the microtiter plate were coated with synthetic CEA instead of eluted TAA. This was considered as a positive control because CEA is a known component of TAA isolated from cancerous cells.
  • the immunized camel serum and passive immunized camel calf serum are far significantly higher than pre-immunized camel serum and pre-immunized camel calf serum respectively (P ⁇ 0.00001). Furthermore, immunized camel serum and passive immunized camel calf serum are far significantly higher than blocking buffer (P ⁇ 0.00001). Meanwhile, no significant differences were between in blocking buffer, pre-immunized camel calf serum, and pre- immunized camel serum ( Figure 1).
  • OD values of serum, milk, and urine samples were significantly higher than negative controls (A, B, and C) (P ⁇ 0.0001).
  • AML showed high variation in the level of anti-TAA antibodies among serum, milk, and urine. It was found that urine revealed highest level of anti-TAA antibodies, followed by serum and then by milk (P ⁇ 0.01 ) ( Figure 2).
  • a L antibodies concentration of lgG2 from serum, milk, and urine samples were significantly higher than negative controls (A and B) (P ⁇ 0.0001).
  • the level of anti-TAA lgG2 antibodies from serum, milk, and urine samples in AML was non-significantly different from that in CLL (P: 0.63, 0.08, 0.7, respectively).
  • lgG2 antibodies level from serum, milk, and urine samples were insignificantly different from that of positive control, CEA (P: 0.85, 0.37, 0.11 , respectively).
  • CEA positive control
  • AML showed no variation in the level of anti-TAA antibodies among serum, milk, and urine (P> 0.01) ( Figure 3).
  • the invention has also shown that the adult camel's urine contains higher anti-CEA IgG than adult camel's serum.
  • the promising result of this phase is that the anti-CEA IgG antibodies were most abundant in urine where is the easiest fluid collected from the animal. Collecting urine does not need invasive injections, like in serum case, or restrict the yield period to the post-pregnancy time, like in milk case.
  • this is the first time to find huge amount of immunoglobulin in urine whether from any animal or human. Therefore, this invention will make the urine of camel as important source of IgG that means the camelid urine becomes the inexpensive valued immunotherapeutic solution and this is an interesting finding.
  • camel's milk contains higher amount of IgGs than that of serum but did not reach significance level.
  • the invention has shown that the CEA- IgG is present in high level in the calf serum passed via udder feeding from the immunized mother camel. Moreover, the significant breakthrough that there is no difference in the level of anti-CEA IgG in adult and calf sera. This finding confirmed that there were a complete passive immunization of antibodies from mother to its calf occurred through milk feeding. Thus in the similar way, it is now possible to immunize human or huge number of animal herd through-out simple milk feeding from immunized camel against any desired pathogenic antigen, therefore this significantly can cut the cost of expensive immunization of each individual animal and it can be accomplished also within a shorter period. This need further investigation to find-out if immunized camel's milk can passive immunity to other animal species.
  • camel species as a targeted IgG antibodies factory for any desired antigen and easily can use camel milk and in particular the camel urine, which is daily extracted, as a source of this valuable cure.
  • camel's factories against these TAA.
  • the preparation of endless in vitro source of the produced target IgG antibodies via molecular cloning of competent E. coli without using any more serum, milk or urine of the camel. Nevertheless, camel's milk and urine are considered a cheaper and simpler method for neutriceutical approach for getting high quality, single domain, single chain camelid IgG against any desired pathogenic antigen.
  • camel animals showed unique capability to excrete high amount of TAA-specific IgG antibodies into urine without affecting the functionality of their immune system. This unique feature of camels that are not seen in other animals renders them a feasible source of specific, robust and smart immunotherapy.
  • CEA carcinoembryonic antigen
  • Tumour stroma-associated antigens for anti-cancer immunotherapy Tumour stroma-associated antigens for anti-cancer immunotherapy.
  • an immunotoxin such as a mAb-ricin A-chain (RA) conjugate rather than un-conjugated mAbs
  • IT immunotoxin
  • the mAb acts primarily as the specific delivery vehicle and RA acts as the cytotoxic effector.
  • the enzymatically active RA is an extremely potent cytotoxic agent, when introduced into the cytoplasm of cells that acts catalytically to inhibit protein synthesis (Seon 1984; Tebbi et al. 1985; Hara and Seon 1987; Hara et al.
  • Immunotoxin is composed of a targeting moiety, such as a ligand or an antibody that has cell type selectivity, linked to a protein toxin with extraordinary potency (Baluna and Vitetta 1997).
  • the targeting moiety recognizes and delivers the whole molecule to the specific receptors on cell surface of the malignant cells.
  • the toxin then triggers cell death either by reaching the cytosol and catalytically inactivating vital cell processes or by modifying the tumor cell membrane.
  • Immunotoxins can be divided into two categories: chemical conjugates (or first-generation immunotoxins) and recombinant immunotoxins (or second-generation immunotoxins), both of which contain a toxin with either mutated or deleted cell-binding domain that prevents it from binding to normal cells (Seon 1984).
  • the truncated toxin can be either fused or chemically conjugated to a ligand or an antibody specific for cancer cells (Li, et al. 2005; Kreitman 2006).
  • the binding of the targeting ligand with these receptors is essentially the first step for these immunotoxins to exert their cytotoxic effect (Allen 2002). Since the target molecules for many toxins are located in the cytosol, receptor-mediated endocytosis is essential for the toxins to perform cytotoxicity. Since transmembrane domain has been removed from most toxin moieties of immunotoxins, the targeting moiety is required to be able to mediate internalization of immunotoxins upon the binding to targets that are present in large amounts on cancer cells. If the ligand-receptor complex is unable to be internalized, the immunotoxin is likely to have a low activity (Allen 2002). Once the complex enters the cytosol, the toxin is then released to inhibit protein synthesis and kills the cells (Li et al. 2005).
  • the immunotoxins were prepared by conjugating ricin A chain (RA) with the camelid antibodies.
  • the ascitic human T-cell leukemia cell tumours were generated via intraperitoneal (i.p.) into nude mice by transplanting Ichikawa cells (a human T-cell leukemia cell line).
  • Ichikawa cells a human T-cell leukemia cell line.
  • mice Each group of 10 mice was injected with one of the following mixtures: (i) 40 pg of purified control mouse IgG (group 1), (ii) 40 g of control RA conjugate (control mouse IgG -RA) (group 2), (iii) 40 g of purified camelid leukemia TAA-specific IgGs (group 3), or (iv) with 40 [ig of purified camelid leukemia TAA-specific IgGs-RA conjugates (group 4).
  • camelid anti-T-cell leukemia antibodies are capable of completely suppressing the tumour growth of human T-cell leukemia cells without any distinct undesirable toxicity. It was also shown that these camelid antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells.
  • mice of groups 1 and 2 formed large ascitic tumors, and all died between 5-7 weeks after the transplantation of the Ichikawa cells.
  • the average survival time of group 1 mice treated with control IgG was 44.5 days and that of group 2 mice treated with control RA conjugate was about the same.
  • Treatment of tumor-bearing mice with purified camelid TAA IgGs (group 3) extended their average survival time only slightly, 5.5 days, and all 10 mice died 6-8 weeks after transplantation of the Ichikawa cells.
  • none of the group 4 mice that were transplanted with Ichikawa cells and treated with the specific anti-T-cell leukemia purified camelid TAA IgGs-RA conjugates developed any tumor, and they were indistinguishable from healthy normal nude mice.
  • the special complete medium was used to isolate and culture of the leukemia cells as the following:
  • Fresh heparenized blood was diluted 1 :1 in RPM1 1640 or PBS; mixed gently until they had been homogenous.
  • the cell suspension was centrifuged in cooling centrifuge (at 4 C 2000 rpm for 10 min) after mixing with equal volume of Ficoll-Paque; the mid layer which contains cells was taken while the supernatant was discarded.
  • Cells were washed three times in phosphate- buffered saline (PBS), and the cells were re-suspended in the complete medium. Cells were counted using haemocytometer Neubaur chamber.
  • the complete growth medium which is used to culture leukemia cells was supplemented with the fetal calf serum 10% (FCS), the growth factors that include human Granulocyte-Macrophage Colony Stimulating Factor(GM-CSF 20 ng ⁇ ml) and G-CSF (20 ng ⁇ ml), and the DMSO 5% as cryopreservation material and the conditioned medium.
  • FCS fetal calf serum 10%
  • GM-CSF 20 ng ⁇ ml human Granulocyte-Macrophage Colony Stimulating Factor
  • G-CSF 20 ng ⁇ ml
  • DMSO 5% cryopreservation material
  • the cells were cultured on the 4 wells micro titer plates. The cells count was about 10 6 cells in each well. Plates were incubated in C02 incubator at 37 C for at least 7 days. A negative control was run with the leukemia culture this was separated from the normal healthy person's peripheral blood.
  • the leukemia culture was divided into different plates according to
  • the leukemia's cases were ALL, CLL and CML and they were different from the cases that the camelid TAA IgG were isolated from to confirm they are cancer specific but not individual specific.
  • the tissue culture was examined for the effect of the formula added and for the presence of the blast cells that characterize the leukemia's cells and blood film stained by special stain like lieshman's stain was carried out to do so. Also the cell count of the tissue culture was examined by using trypan blue- Neubaur hemoctymeter chamber.
  • mice Seven- to 8-week of age female BALB/c (nu/nu) mice were obtained from Experimental Therapeutic Department/the National Center of the Cancer and Genetic Research (Bagdad Iraq). The mice were maintained in the central animal house under sterile conditions in cages with filter bonnets in a laminar flow unit (Lab Products, Maywood, NJ). They were given sterilized pellet food and tap water.
  • lchikawa a human T-cell leukemia cell line was kindly provided by Experimental therapeutic Department the National Center of the Cancer and Genetic Research (Bagdad Iraq), lchikawa was cultured in RPMI 1640 medium supplemented with 4% (vol/vol) heat-inactivated fetal calf serum. as described (Seon 1984; Hara and Seon 1987; Hara et al. 1988). Reagents
  • Purified camelid TAA IgGs were conjugated with RA (cytotoxic effector) using N-succinimidyl-3-(2-pyridyldithio) propionate (Seon 1984; Tebbi et al. 1985; Hara and Seon 987; Hara et al. 1988; Li et al. 2005).
  • Purified IgGs in PBS (0.01 sodium phosphate, pH 7.4, containing 0.15 M NaCI) were treated with a 15-fold molar excess of N-succinimidyl-3-(2-pyridyldithio) propionate for 30 min at room temperature to introduce 2-pyridyl disulfide groups into the IgG molecule.
  • the modified and dialyzed proteins were then mixed with a 3-fold molar excess of the purified, freshly reduced RA in PBS containing 1 mM EDTA and incubated at 40C for 15 hr.
  • the IgG-RA conjugates were separated from the unbound RA by gel filtration on a calibrated Sephacryl S-200 column. The conjugates were stored at -80°C after penicillin (100 units/ ml), streptomycin (100 ug/ml), and gentamicin (50 ug/ml) were added.
  • the lchikawa cell suspension (1 ml with 1.6 x 10 7 cells) in saline was injected i.p. into a nude mouse to form an ascitic tumor (Seon 1984; Tebbi et al. 1985; Hara and Seon 1987; Hara et al. 1988; Li et al. 2005). All 35 of the 35 mice injected developed ascitic tumors, lchikawa cells were successfully transplanted into nude mice and formed ascitic tumors. Treatment of Ascitic Tumor with Cytotoxic Conjugates
  • a nude mouse was injected i.p. with PBS containing 40 pg of cytotoxic conjugates (CCs) or with 40pg of control conjugates 2 hr after the injection of lchikawa cells. The injection of CCs was repeated on day 4. In the subsequent experiment, four groups of nude mice were injected i.p.
  • CCs cytotoxic conjugates
  • the cell count was carried out 3 days after addition of camelid TAA IgG product and cytotoxic conjugate.
  • the cultured cells were checked daily to observe the cells and premature cells (Blasts).
  • the cells count results were excelled and the blast cells were stained by lishman stain to read cells film.
  • mice of groups 1 and 2 formed large ascitic tumors, and all died between 5.0-7.0 weeks after the transplantation of the Ichikawa cells.
  • the average survival time of group 1 mice treated with control IgG was 44.5 days and that of group 2 mice treated with control RA conjugate was about the same.
  • results obtained using the ascitic tumour system are remarkably good in that purified camelid IgGs -RA completely suppressed the tumour growth of human T-cell leukemia cells without showing any distinct toxicity in all nude mice tested, whereas control RA conjugate was not effective at all in suppressing the tumour.
  • results indicate a strong potential of purified camelid IgGs -RA for treating T-cell leukemia patients.
  • the data presented in this invention provide strong evidence that ITs indeed have good potential for in vivo therapy of human diseases if purified camelid TAA IgGs are used in preparing the ITs in which the selectivity can be improved.

Abstract

La présente invention concerne un procédé pour la production d'immunoglobuline de liaison spécifique aux cellules cancéreuses, ledit procédé comprenant l'immunisation d'un camélidé avec un antigène associé aux tumeurs purifié dérivé de cellules cancéreuses par élution à l'acide et la purification d'immunoglobuline spécifique de l'antigène associé aux tumeurs dérivé d'un liquide organique dudit camélidé. L'invention concerne également des immunoglobulines de camélidés spécifiques des cellules cancéreuses produites par le procédé selon l'invention et des procédés pour le traitement ou la prévention du cancer comprenant l'administration desdites immunoglobulines spécifiques des cellules cancéreuses, ou des immunotoxines qui en sont dérivées.
PCT/GB2011/050382 2010-02-25 2011-02-25 Anticorps de camélidés destinés à être utilisés dans des compositions et procédés pour le traitement du cancer WO2011104565A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140072649A1 (en) * 2012-09-11 2014-03-13 Al-Urdonia Lemudaddat Al-Ajsam Co. Immunized Camel Milk-Based Composition for the Treatment or Prevention of Gastrointestinal Infections
US20140072648A1 (en) * 2012-09-11 2014-03-13 Khaled Mahmood Al-Qaoud Camel Milk-Based Topical Pharmaceutical Composition
WO2018140525A1 (fr) * 2017-01-24 2018-08-02 Abexxa Biologics, Inc. Méthodes et compositions destinées à cibler un complexe comprenant un hla-i non classique et un néo-antigène dans le traitement du cancer
WO2021116699A1 (fr) 2019-12-11 2021-06-17 Precision Immunotherapeutics Limited Molécules de liaison inhibées de manière réversible

Non-Patent Citations (48)

* Cited by examiner, † Cited by third party
Title
ALLEN TM.: "Ligand-targeted therapeutics in anticancer therapy", NAT REV CANCER, vol. 2, 2002, pages 750 - 763
BALUNA R, VITETTA ES.: "Vascular leak syndrome: a side effect of immunotherapy", LMMUNOPHARMACOLOGY, vol. 37, 1997, pages 117 - 132, XP002585736
BOON, T., CEROTTINI, J. C., VAN DEN EYNDE, B., VAN DER BRUGGEN, P., VAN PEL, A.: "Tumour antigens recognized by T lymphocytes", ANNU REV IMMUNOL., vol. 12, 1994, pages 337 - 365
CHEEVER, M. A., CHEN, W., DISIS, M. L., TAKAHASHI, M., PEACE, D. J.: "T-cell immunity to oncogenic proteins including mutated ras and chimeric bcr-abl", ANN N Y ACAD SCI., vol. 690, 1993, pages 101 - 112
CROCE, C. M.: "Oncogenes and cancer", N ENGL J MED., vol. 358, 2008, pages 502 - 511
CURIEL T.J.: "Tregs and rethinking cancer immunotherapy", J CLIN INVEST., vol. 117, 2007, pages 1167 - 1174
DESMYTER, A., TRANSUE, TR., ARBABI GHAHROUDI, M., DAO-THI, M., POORTMANS, F., HAMERS, R., MUYLDERMANS, S., WYNS, L.: "Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme", NATURE STRUCTURAL BIOLOGY, vol. 3, 1996, pages 803 - 811
FAY, J.W., MOORE, J.O., LOGUE, G.L., HUANG, A.T.: "Leukopheresis therapy of leukaemic reticuloendotheliosis (hairy cell leukaemia)", BLOOD, vol. 54, 1979, pages 747 - 749
FISK, B, BLEVINS, T. L., WHARTON, J. T., OANNIDES, C. G.: "Identification of an immunodominant peptide of HER-2/neu proto-oncogene recognized by ovarian tumour-specific cytotoxic T lymphocyte lines", J EXP MED., vol. 181, 1995, pages 2109 - 2117
GERMAIN, R.N., MARGULIES D.H.: "The biochemistry and cell biology of antigen processing and presentation", ANNU REV IMMUNOL., vol. 11, 1993, pages 403 - 450
GRITZAPIS, A.D., SOTIRIADOU, N.N., PAPAMICHAIL, M., BAXEVANIS, C.N.: "Generation of human tumor-specific CTLs in HLA-A2.1-transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors", CANCER LMMUNOL LMMUNOTHER, vol. 53, 2004, pages 1027 - 1040
HALL WA, FODSTAD O.: "Immunotoxins and central nervous system neoplasia", J NEUROSURG, vol. 76, 1992, pages 1 - 12
HAMERS-CASTERMAN, C., ATARHOUCH, T., MUYLDERMANS, S., ROBINSON, G., HAMERS, C., BAJYANA SONGA, E., BENDAHMAN, N., HAMERS, R.: "Naturally occurring antibodies devoid of light chains", NATURE, vol. 363, 1993, pages 446 - 448, XP002535892, DOI: doi:10.1038/363446a0
HAMMARSTROM S.: "The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues", SEMIN CANCER BIOL, vol. 9, 1999, pages 67 - 81
HARA H, LUO Y, HARUTA Y, SEON BK: "Efficient Transplantation of human non-T-leukemia cells into nude mice and induction of complete regression of the transplanted distinct tumours by ricin A-chain conjugates of monoclonal antibodies SN5 and SN61", CANCER RESEARCH, vol. 48, 1988, pages 4673 - 4680
HARA H, SEON BK: "Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: Nude mouse models", PROC. NATI. ACAD. SCI. USA, vol. 84, 1987, pages 3390 - 3394
HOFMEISTER, V., VETTER, C., SCHRAMA, D., BROCKER, E. B., BECKER, J. C.: "Tumour stroma-associated antigens for anti-cancer immunotherapy", CANCER IMMUNOL LMMUNOTHER., vol. 55, 2006, pages 481 - 494, XP019333238, DOI: doi:10.1007/s00262-005-0070-1
JASSIM, S.A.A., NAJI, M.A.: "The desert ship heritage and science", BIOLOGIST (UK), vol. 48, 2001, pages 268 - 272
KREITMAN RJ, PASTAN: "Immunobiological treatments of hairy-cell leukaemia", BEST PRACT RES CLIN HAEMATOL., vol. 16, 2003, pages 117 - 133
KREITMAN RJ.: "Immunotoxins for targeted cancer therapy", THE AAPS JOURNAL, vol. 8, no. 3, 2006, pages E532 - E551
KUTVIRT, S. G., LEWIS, S. L., SIMON, T. L.: "Lymphocyte phenotypes in infants are altered by separation of blood on density gradients", BR J BIOMED SCI., vol. 50, 1993, pages 321 - 328
LASKOWSKI, R.A., LUSCOMBE, N.M., SWINDELLS, M.B., THORNTON, J.M .: "Protein clefts in molecular recognition and function", PROTEIN SCI., vol. 5, 1996, pages 2438 - 2452, XP001030536
LATEEF, S. S., GUPTA, S., JAYATHILAKA, L. P., KRISHNANCHETTIAR, S., HUANG, J. S., LEE, B. S.: "An improved protocol for coupling synthetic peptides to carrier proteins for antibody production using DMF to solubilize peptides", J BIOMOL TECH., vol. 18, 2007, pages 173 - 176
LAYNE E: "Methods I Enzymology", vol. ILL, 1957, ACADEMIC PRESS, article "Spectrophotometric and turbidimetric methods for measuring proteins", pages: 447 - 455
LI Z, YU T, ZHAO P, MA J.: "Immunotoxin and cancer therapy", CELLULAR AND MOLECULAR IMMUNOLOGY J, vol. 2, 2005, pages 106 - 112
MALYANKAR, U. M.: "Tumour-associated antigens and biomarkers in cancer and immune therapy", INT REV IMMUNOL., vol. 26, 2007, pages 223 - 247
MUNA E AHMED ET AL: "Bacteria Associated with Healthy Sudanese Camels' Urine and Drug Susceptibility of some Bacteria of Human Origin to Camel Urine", INTERNET CITATION, pages 2PP, XP009148215, ISSN: 1028-9437, Retrieved from the Internet <URL:http://sudanjvr.net/10-23.html> [retrieved on 20110511] *
NIJMAN, H. W., VAN DER BURG, S. H., VIERBOOM, M. P., HOUBIERS, J. G., KAST, W. M., MELIEF, C. J.: "p53, a potential target for tumour-directed T cells", IMMUNOL LETT., vol. 40, 1994, pages 171 - 178
OMIDFAR K ET AL: "PRODUCTION AND CHARACTERIZATION OF A NEW ANTIBODY SPECIFIC FOR THE MUTANT EGF RECEPTOR, EGFRVIII, IN CAMELUS BACTRIANUS", TUMOR BIOLOGY, KARGER, BASEL, CH, vol. 25, no. 4, 1 January 2004 (2004-01-01), pages 179 - 187, XP009044126, ISSN: 1010-4283, DOI: DOI:10.1159/000081100 *
OMIDFAR K ET AL: "Production of a novel camel single-domain antibody specific for the type III mutant EGFR", TUMOR BIOLOGY, KARGER, BASEL, CH, vol. 25, no. 5-6, 1 September 2004 (2004-09-01), pages 296 - 305, XP009147922, ISSN: 1010-4283, DOI: DOI:10.1159/00081395 *
OSTANKOVITCH, M., LE GAL, F.A., CONNAN, F., CHASSIN, D., CHOPPIN, J., GUILLET, J.G.: "Generation of Melan-A/MART-1 -specific CD8+ CTL from human naive precursors: Helper effects requirement for efficient in vitro primary CTL induction", INT J CANCER., vol. 72, 1997, pages 987
PENNELL CA, ERICKSON HA.: "Designing immunotoxins for cancer therapy", LMMUNOL RES, vol. 25, 2002, pages 177 - 191
RAMMENSEE, H.G., FALK, K., ROTZSCHKE, O.: "Peptides naturally presented by MHC class I molecules", ANNU REV IMMUNOL., vol. 11, 1993, pages 213 - 244, XP002912058, DOI: doi:10.1146/annurev.iy.11.040193.001241
REE, A. H., BRATLAND, A., DUELAND, S.: "Molecular targeted therapy in colorectal cancer", TIDSSKR NOR LAEGEFOREN, vol. 128, 2008, pages 190 - 193
REITER Y, PASTAN: "Recombinant Fv immunotoxins and Fv fragments as novel agents for cancer therapy and diagnosis", TRENDS BIOTECHNOL, vol. 16, 1998, pages 513 - 520, XP004143812, DOI: doi:10.1016/S0167-7799(98)01226-8
REITER Y.: "Recombinant immunotoxins in targeted cancer cell therapy", ADV CANCER RES, vol. 81, 2001, pages 93 - 124
SEON BK: "Specific killing of human T-Leukemia cells by immunotoxins prepared with ricin A chain and monoclonal anti-human T-Cell leukemia antibodies", CANCER RESEARCH, vol. 44, 1984, pages 259 - 264
SHEBZUKHOV LU, V., BELOUSOV, P. V., KHLGATIAN, S. V., SAZYKIN, A., KUPRASH, D. V., NEDOSPASOV, S. A.: "Contemporary molecular-biological methods of cancer diagnosis and monitoring based on the humoral immune response to tumour-associated antigens", MOL GEN MIKROBIOL VIRUSOL., vol. 4, 2007, pages 3 - 6
SLOCUM, H. K., PAVELIC, Z. P., RUSTUM, Y. M., CREAVEN, P. J., KARAKOUSIS, C., TAKITA, H., GRECO, W. R.: "Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors", CANCER RES., vol. 41, 1981, pages 1428 - 1434, XP000887063
STORKUS, W.J., ZEH, H.J.I., SALTER, R.D.: "Identification of T-cell epitopes: rapid isolation of class I-presented peptides from viable cells by mild acid elution", J LMMUNOTHER., vol. 14, 1993, pages 94 - 103, XP009009060
TEBBI C, CHERVINSKY T, SEON B: "Effect of specific anti-human leukemia immunotoxins on the colony formation by human haematopoietic progenitors and by human leukemia cells", CLIN. EXP. LMMUNOL, vol. 60, 1985, pages 496 - 500
URBAN, J.L., SCHREIBER, H.: "Tumour antigens", ANNU REV LMMUNOL., vol. 10, no. 22, 1992, pages 617 - 644
VALSECCHI, M. G., STELIAROVA-FOUCHER, E.: "Cancer registration in developing countries: luxury or necessity?", LANCET ONCOL., vol. 9, 2008, pages 159 - 167, XP022479876, DOI: doi:10.1016/S1470-2045(08)70028-7
VIRNA, C-R., BACKMANN, N., SENTER, P.D., WERNERY, U., BAETSELIER, PD, MUYLDERMANS, S., REVETS, H.: "Efficient Cancer Therapy with a Nanobody-Based Conjugate", CANCER RESEARCH, vol. 64, 2004, pages 2853 - 2857, XP002360683, DOI: doi:10.1158/0008-5472.CAN-03-3935
WANG RONG-FU: "Human tumor antigens: Implications for cancer vaccine development", JOURNAL OF MOLECULAR MEDICINE (BERLIN), vol. 77, no. 9, September 1999 (1999-09-01), pages 640 - 655, XP002637325, ISSN: 0946-2716 *
WEBSTER, D.M., HENRY, A.H., REES, A.R.: "Antibody-antigen interactions", CURR OPIN STRUCT BIOL., vol. 4, 1994, pages 123 - 129
WU HERREN ET AL: "Cloning, isolation and characterization of human tumor in situ monoclonal antibodies", CANCER IMMUNOLOGY IMMUNOTHERAPY, vol. 51, no. 2, April 2002 (2002-04-01), pages 79 - 90, XP002260156, ISSN: 0340-7004 *
YOSHINO, I., GOEDEGEBUURE, P.S., PEOPLES, G.E.: "HER2/neu-derived peptides are shared antigens among human non-small lung cancer and ovarian cancer", CANCER RES., vol. 54, 1994, pages 3387 - 3390, XP001204364

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140072649A1 (en) * 2012-09-11 2014-03-13 Al-Urdonia Lemudaddat Al-Ajsam Co. Immunized Camel Milk-Based Composition for the Treatment or Prevention of Gastrointestinal Infections
US20140072648A1 (en) * 2012-09-11 2014-03-13 Khaled Mahmood Al-Qaoud Camel Milk-Based Topical Pharmaceutical Composition
US10010564B2 (en) * 2012-09-11 2018-07-03 Khaled Mahmood Al-Qaoud Camel milk-based topical pharmaceutical composition
US10278404B2 (en) * 2012-09-11 2019-05-07 Al-Urdonia Lemudaddat Al-Ajsam Co Immunized camel milk-based composition for the treatment or prevention of gastrointestinal infections
WO2018140525A1 (fr) * 2017-01-24 2018-08-02 Abexxa Biologics, Inc. Méthodes et compositions destinées à cibler un complexe comprenant un hla-i non classique et un néo-antigène dans le traitement du cancer
WO2021116699A1 (fr) 2019-12-11 2021-06-17 Precision Immunotherapeutics Limited Molécules de liaison inhibées de manière réversible

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