WO2016081889A1 - Recombinant c1 esterase inhibitor and use thereof - Google Patents
Recombinant c1 esterase inhibitor and use thereof Download PDFInfo
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- WO2016081889A1 WO2016081889A1 PCT/US2015/061967 US2015061967W WO2016081889A1 WO 2016081889 A1 WO2016081889 A1 WO 2016081889A1 US 2015061967 W US2015061967 W US 2015061967W WO 2016081889 A1 WO2016081889 A1 WO 2016081889A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
- G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
Definitions
- the present invention relates to a method a method for the production of recombinant CI esterase inhibitor (CI IA), a human host cell encoding the CI esterase inhibitor (CI IA), a recombinant CI esterase inhibitor (CI IA), a composition comprising the recombinant CI esterase inhibitor (CI IA), use of the recombinant CI esterase inhibitor (CI IA), an antibody raised against the recombinant CI esterase inhibitor (CI IA), a method for detecting and/or screening and/or monitoring cancer in an individual and a method of diagnosing rejection of transplants as well as a method using a human or humanized antibody raised against the recombinant CI esterase inhibitor for repeated treatment of certain cancer types with said antibody.
- an object of the present invention relates to the surprising findingCl esterase inhibitor (CI IA) and native C4 can be used in a method for detecting and/or screening and/or monitoring cancer in an individual.
- one aspect of the invention relates to a method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines.
- CI IA CI esterase inhibitor
- Another aspect of the present invention relates to a method for producing recombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI esterase inhibitor developed at the end of the Fab or (Fab) 2 of the immunoglobulin.
- the anti CI esterase inhibitor "paratope” (analogous to a lock), which is located at each tip of the "Y" of an antibody. This paratope is specific for one
- a further aspect the present invention pertains to a composition comprising a recombinant CI esterase inhibitor, characterised in that
- composition comprises substantially no synthetic protease inhibitors, and/or
- composition is serum free.
- present invention pertains to a recombinant CI esterase for use as a medicament.
- the present invention pertains to a method for preventing, treating and/or alleviating Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI esterase inhibitor.
- Ischemic stroke reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation
- the present invention pertains to a method for treating burn wounds, Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor.
- the invention pertains to an antibody raised against a CI esterase inhibitor.
- the invention pertains to a method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
- CI IA inactivator
- second parameter represented by the concentration of complement component native C4 in at least one excreta from the individual wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
- the invention pertains to a kit for performing the above method, wherein said kit comprises CI IA and native C4.
- the invention pertains to a method of diagnosing rejection of transplants, said method comprising the steps of
- the invention pertains to a method of diagnosing cancer, said method comprising the steps of
- the most important goal in the opinion of the inventor is, to identify, develop, and produce an efficient antidote to neutralize CI IA coat and at the same time balance the treatment in such a manner that the necessary amount of CI IA in circulating blood, the lymphatic system and other body fluids including the necessary amount of CI IA in the CNS system to avoid the treatment to close down the CI IA production to a level, where the "classical pathway" for complement can still be active in order to avoid that the patient develop an angioedema also called HANE-like condition.
- biopharmaceutical or other biological product - approaches that can inhibit CI IA or its inhibitory effect.
- Aprotinin is not in the focus as an inhibitor of CI inactivator, and even though Aprotinin is a xenogeic protein, there is a possibility that those researchers working with Aprotinin might produce a recombinant anti Aprotinin that in theory could inhibit CI inactivator activity; for instance Wachtfogel et al described that for instance Kallikrein-Cl-inhibitor complexes were completely inhibited and Cl-Cl-inhibitor complexes were partially inhibited
- Such approach could in theory be approached by obtaining blood from a patient with angioedema associated with a B-celllymphoproliferative disorder that became evident 9 months after C1INH deficiency was diagnosed, and androgen therapy stopped the attacks of angioedema.
- This category of patients with lymphoproliferative disease and angioedema has been described previously (Frigas E. Mayo Clin Proc. 1989 Oct;64(10) : 1269-75). Cicardi et al found at the department of Internal Medicine, University of Milan, Italy, among 23 patients through some years with angioedema.
- Autoantibodies against CI inactivator was present in 17 of these patients, and in some of these patients were 3 patients with (Non Hodgkins) lymphomas, and one patient with chronic lymphatic leukemia (Cicardi M, Zingale LC, Pappalardo E, Folcioni A, Agostoni A. Autoantibodies and
- B lymphocytes could be harvested from for instance 50 cc of fresh blood, B lymphocytes (whereof some of these will produce anti CI inactivator) could be isolated by using Hypaque Ficoll centrifugation. These B lymphocytes could be cultured and the anti CI inactivator secreting B lymphocytes could be caught by immobilized CI inactivator using a cell sorting equipment. These B cells could then be proliferated and the immunoglobulin producing antigenic determinants against CI inactivator could be isolated, and the antigenic determinants of these immunogloubulins could be subjected to molecular biological isolation of the antigenic determinants, and the code for the anti CI inactivator could be disclosed.
- This code could then be the basis for the production of recombinant human IgG (or IgM) with binding specificity to Clinactivator.
- a human recombinant anti human CI inactivator which could isolated, purified and used for the infusions in humans without risking any significant allergic or immune reaction in patients.
- cancer patients with carcinoma cells or other malignant cells with CI inactivator coat could then be treated repeatedly, and of course monitored by measuring the blood CI inactivator and C4, to balance the treatment with anti CI inactivator by following the balance, so that the patient can be safely treated without falling below the until further postulated low levels of blood CI inactivator and blood C4.
- the theoretically most ideal antibody would be a human or humanized anti human CI IA antibody that at the same time can neutralize the CI IA activity and secondly alert the initiated antigen - antibody reaction against CI IA at the same time could activate the "classical pathway" of complement, meaning an immunoglobulin of type as for instance IgG (minus IgG 4 ) or IgM that at the same time would cause disassociation of the Clqrs complex to a degree, where C4 is activated and start the binding to the surface of the cell which again would activate C3, C5 all the way to C9 where these cumulative bindings would end up producing "pores" on the cancer cell and thereby lysing the cell.
- an immunoglobulin of type as for instance IgG (minus IgG 4 ) or IgM that at the same time would cause disassociation of the Clqrs complex to a degree, where C4 is activated and start the binding to the surface of the cell which again would activate C3, C5 all the way to C9 where these cumulative bindings
- the antibody most ideal for this purpose would also be capable of being repeatedly infused to the patient during months may be years to obtain the most efficient kill of the cancer cells.
- Another approach of the specific human, preferably recombinant or humanized monoclonal antibody with high binding capacity being capable of carrying either a cancer-killing component, may be in a form of a potent
- chemotherapeutic agent which otherwise could be given systemically without producing potent and serious side effects.
- Another approach could be to bind a radioactive emitter to the cell for direct cell killing amount of radioactive doses, such as for instance a beta emitter with relatively low spreading to surrounding tissue.
- technetium-99 (itself an isotope of technetium), symbolized as 99m Tc, that is used in tens of millions of medical diagnostic procedures annually, making it the most commonly used medical radioisotope.
- Technetium-99m is used as a radioactive tracer and can be detected in the body by medical equipment (gamma cameras).
- CI IA (mammalian) CI inactivator (CI IA) in human antibody, either as humanized monoclonal antihuman CI IA or as human recombinant anti human antibody , as IgG or IgGl or IgG3, to be used parenterally for the treatment of cancer of type Carcinoma, malignant brain tumors such as Astrocytomas/glioblastomas, or certain types of malignant Sarcomas benefitting patients with primary cancers or metastases
- the primary goal of an anti human CI IA antibody is that one can repeat the treatment, unlike what one can do with xenogeneic antibodies, which historically indicated to have tumor regression effect, but at the same time, only academic interest, because one could only administer the xenogeneic antibody once or twice, otherwise the patient showed immunogenic reaction against the xenogeneic antibody, and prevented further treatment even though one could demonstrate effect in regards to tumor regression in patients treated with this anti CI IA.
- a continuous treatment over weeks, months and years can be done, when the human does not produce antibodies to the exogenously applied humanized monoclonal anti CI IA or even better applying a recombinant human anti CI IA (e.g., preferably IgGl) and at the same time follow the decrease of the increased CI IA found in cancer patients circulating in the blood proving to be an excellent way of indicating effect at the same time as the cancer
- Figures 4 and 5 present serum CI IA and serum native C4 (meaning these proteins circulating in the blood) during the treatment of a 28 month old infant with Xenogeneic (porcine) polyclonal anti human semipurified IgG fraction of antibody from pigs immunized with semipurified CI IA.
- Native C4 any other native protein is to be understood as the native state of a protein or nucleic acid which its properly folded and/or assembled form, which is operative and functional.
- Most human cancer types ( ⁇ 85% of all cancers) are carcinomas and these types of cancers have shown the presence of an inhibitor of the complement system, that the inventor have found, is the CI inactivator (also called CI inhibitor or CI esterase inhibitor).
- This type of cancer that appears to exhibit the CI inactivator (C1IA) on their plasma membrane are derived from epithelial cells that arises in the embryon from cell in the endodermal or ectodermal germ layer. It is postulated that these types of cancers occur because of altered DNA in these cells.
- the inventor have also indicated that certain malignant brain tumors such as astrocytomas,
- glioblastomas, medulloblastomas and other types of primary brain tumors by testing some of these primary brain tumors with rabbit anti human CI inactivator-FITC conjugated, also carry this inhibitor.
- the primary malignant brain tumors when cultured in cell culture flasks, have shown that they after one or more sub-culturing processes lose the ability apparently to produce CI inactivator and present this on their plasma membrane. This is significantly different from the carcinomas, which in subcultures repeatedly reproduce the CI inactivator (or CI IA) on their plasma membrane, even when carcinomas have metastasized to the brain (thus being sometimes described as secondary brain cancer).
- the definite difference between the carcinomas and the primary malignant brain tumors is postulated by the inventor to be caused by the fact - that it is known that primary brain tumors (even though they are sufficiently
- the brain tumors are accordingly protected against the complement cascade as long as they are retaining their CI inactivator on their plasma membrane, but are apparently loosing this ability to produce more CI inactivator for protection when sub-cultured, and therefore the primary brain cancer cells will either lack the ability to grow outside the CNS, because of unknown factors keeping them alive in the CNS, or if they are released into the blood
- condition outside the CNS may differentiate the cells as has been observed in the microscope when studying subcultured CNS brain cancer cells, because their appearance change towards resembling fibroblast- like cells and as discussed above, lacking the CI Inactivator coat on their plasma membrane.
- Carcinoma is a type of cancer that develops from epithelial cells.
- a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during embryogenesis.
- Carcinomas are the most common type of cancer. They make up about 85 out of every 100 cancers in the UK (85%).
- anti CI inactivator would first of all according to the inventor be one major important reason for cancer to evade the body's humoral immune defense system (which again normally interact with the cellular immune system at many levels) by simply blocking the complement activation already at the first complement component, the Clqrs complex where the normal dissociation of this complex would result in activation of the full C4
- Cancer in vivo diagnostic and tracer e.g., a radioactive isotope by labelling a humanized antibody against CI inactivator.
- Cancer treatment agent such as anti human or anti humanized CI inactivator 4.
- Cancer human or humanized anti CI inactivator coupled to a anti cancer treatment drug which e.g., could be delivered directly to the target cells
- This invention provides a unique and novel way to side-track the protecting CI inactivator coat, by introducing a recombinant human IgG antibody having antigen binding sites against CI inactivator.
- lymphocytes and for instance by using molecular methods to detect the appearance of the paratope directed against the CI inactivator.
- diseases as acquired angio-oedema; - ymphoproliferative disease;
- CI-INH CI inhibitor
- AAE advanced angio-oedema
- Anti-Cl-INH autoantibodies measured both as immunoglobulin binding to Cl- INH immobilized onto microtitre plates (ELISA) and as plasma inhibitory activity of Cl-INH function, were found in 12 patients. Binding of Cl-INH to paraproteins, transferred to Immobilon after agarose gel electrophoresis, was detectable in five of seven M-components associated with AAE.
- the white blood cells normally work together with the humoral immune system activated complement components to attack any foreign invaders, or foreign cells, so the immune system as such will in its major defence system utilize both the cellular, and the humoral immune system in its elimination of foreign or allogeneic cells.
- carcinoma cells in some way should be postulated to be recognized as “foreign” cells, they are protected to a degree that does not allow the body to get rid of these cells, which I would postulate could be called “protective mutagene” cells, because the difference is most probably a mutation in the DNA which has changed the "protected - normally called malignant cell".
- the body could recognize these cells as "unprotected mutagene cells” instead of protected cancer cells, the body would most probably eliminate these cells that in a non-protective stage via the immunosurveillance system because now the so-called malignant cell (after protection has been stripped from the cell) would be detected as being non-self, and the immune system including the complement system would then eliminate cells as if it was an allogeneic cell, and one could postulate, that if one changes the protective malignant cells and render them unprotected, the body would attack these cells as were they "pseudo-autoimmune cells".
- the cells might be defined as malignant, as long as the body can recognize these cells. May be they should be viewed as a form in a way undistinguishable from "allogeneic" cells that might be protected by some mechanism, which would not recognize them as allogeneic - due to a theoretical protective system, which in theory might appear in the human body.
- malignant could thus appear when the immune system cannot recognize or according to this invention is inhibited from using its immune system fully because of inhibiting factors appearing in the cell that now has turned malignant. So, the malignant cell could also be called a protected cell that can freely spread to any tissue in the body and often. From this view point the malignant cells could also be viewed from the perspective of protection, where the protection is a protein such as CI inactivator, which is not recognized as foreign for the immune surveillance in the body, and therefore, the immune system would not attack the cells using the complement system as eliminator of these cells.
- the protection is a protein such as CI inactivator
- Example 1 and figure 1 the increased circulating CI inactivator and the blocked full C4 component found in figure 1 could explain that cancer patients apparently have increased circulating CI inactivator and full C4 component in the blood. This has to be held together with the findings of CI inactivator coating the cancer cells as shown in Example 2, figure 4.
- a cancer detecting and cancer monitoring kit consisting of highly purified antibodies with high specificity to the CI esterase inhibitor also described as CI inactivator (CI IA) and the and complement component native C4, and certainly not C4a or C4b.
- Native, human C4 complement component is a glycoprotein composed of three non-identical subunits of M.W. 93,000 (a), 75,000 ( ⁇ ), and 32,000 (y) linked by disulfide bonds. Present in normal human serum at 400 pg/ml or 40 mg/100 ml.
- C4a M.W. 8740
- C4b fragments M.W. 193,000.
- the released C4a peptide is one of the three complement-derived anaphylatoxins.
- the nascent C4b fragment can form a covalent ester bond with target surfaces. This covalent attachment of C4b to target acceptors is required for continuation of activation via classical pathway.
- C4a peptide is one of the three complement-derived anaphylatoxins.
- the nascent C4b fragment can form a covalent ester bond with target surfaces. This covalent attachment of C4b to target acceptors is required for continuation of activation via classical pathway.
- carcinomas are the most dominant malignant organ related tumors where the cancer appears to escape the human immune defence system including the complement system, where the classical pathway for a complement cascade, normally related to an antigen-antibody reaction on the surface of a pathological cell or microorganism.
- the dual kits for routine testing will be using two different methods, one being a measurement using immune testing apparatus, such as for instance ELISA readers using conugated recombinant anti human CI IA, conjugated recombinant human C4 or conjugated recombinant C4d for example using Thermo Scientific ELISA equipment.
- immune testing apparatus such as for instance ELISA readers using conugated recombinant anti human CI IA, conjugated recombinant human C4 or conjugated recombinant C4d for example using Thermo Scientific ELISA equipment.
- the other complement component to be measured in the cancer diagnostic kit according to this invention is the "intact" molecule, the native C4. If both CI IA and C4 is elevated, it is according to this invention indicating the presence of active malignant cancer tissue.
- the subunits C4a and C4b does not play a role in this diagnostic kit, but would be more aimed at measuring C4b or other subunits in patients with autoimmune disorders (Mortensen S, Kidmose RT, Petersen SV, Szilagyi A, Prohaszka Z, Andersen GR, J Immunol. 2015 Jun l; 194( l l) : 5488-96).
- C4d Another subunit of C4 is called C4d is reported to be increased in tissue from liver transplant patients showing signs of acute liver rejection or ongoing full acute or chronic liver rejection, so in such case to evaluate the rejection mode in the organism, for instance liver rejection, a C4d measurement would be useful (Gierej B, Kobryh K, Gierej P, Gornicka B, Ann Transplant. 2014 Aug 1; 19 : 373-81).
- a dual cancer diagnostic kit could be to identify any probably significant rejection of carcinoma or metastases thereof, theoretically be another combination of cancer related test kit, namely to detect a decreasing level of CI IA during treatment with infusion of for instance recombinant anti human CI IA, which is the focus of this invention - a breakdown of carcinoma or other cancer tissue, evidenced by a decreasing CI IA, and an elevated C4d subunit. Therefore, a second specific test kit aimed at monitoring cancer cell lysis based on the treatment with infusion of antihuman CI IA antibody, or may be even other form of immune treatment that would cause rejection of cancer cells.
- test kit could according to the inventor be an important tool to monitor possible rejection of cancer, and may be also for monitoring of quite another form of rejection, namely detection of rejection occurring after allogenic organ - or bone marrow transplantation, where most probably, besides the increased activity of the classical complement cascade pathway, For instance indicated by a decreased CI IA and an increased native C4d unit.
- cancer diagnostic kit could consist of measurement of CI IA (to identify a possible decrease) held together with an increased
- the object of the present invention relates to the unique CI IA coating of certain human cancers, especially carcinoma, certain sarcomas and primary malignant brain tumors. These types of malignant tumors represent the most dominant malignant organ related types of tumors, where these cancer types appear to escape the human immune defence system including the
- the equipment used for the measurement of CI IA on carcinoma cells and malignant brain tumor cells as well as measurement of benign cells all harvested from patients by either biopsies from the tumor or by pleura or ascites effusion from patients with metastatic carcinomas.
- the cells were cultured on sterile micro-slides in Leighton tubes and a corresponding NUNC culture flask as monolayer cultures in MEMF12 medium at Danish laboratories in 37°C C0 2 incubators.
- the inventor is aware of the complexity of the various forms of complement components and what and how the various parts or factors in the complement system which is activated and in what order.
- the complement system consists of a number of small proteins found in the blood, in general synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex
- the complement system is a part of the immune system that helps or complements the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the innate immune system, which is not adaptable and does normally not change over the course of an individual's lifetime.
- the complement system is activated when for instance adaptive immune is initiated. There are three pathways through which the complement system can be activated.
- CI inhibitor or CI inactivator
- Cl-inhibitor Cl-inh, CI esterase inhibitor
- CI IA Cl-inhibitor
- Its main function is the inhibition of the complement system to prevent spontaneous activation where the ultimate end point of a classical pathway is cell lysis.
- the "lectin pathway” is a type of cascade reaction in the complement system, similar in structure to the classical complement pathway proceeding directly through the action of C4 and C2 to produce activated complement proteins further down the cascade. In contrast to the classical complement pathway, the lectin pathway does not recognize an antibody bound to its target.
- the lectin pathway starts with mannose-binding lectin to certain glycoproteins of certain microorganisms. This pathway consists of lectin binding to mannose, or glucose determinants on glycoprotein components on microorganisms such as salmonella, listeria, and neisseria, fungal pathogens, and certain types of virus such as HIV-1 and RSV.
- the "alternative pathway” is one of the three complement pathways that opsonize and kill pathogens.
- the pathway is triggered by hydrolysis of complement component 3 when the C3b protein directly binds the microbe.
- the classical pathway of a complement cascade is normally related to an adapt immune reaction such as an antigen-antibody reaction in which IgG or IgM is involved in the antigen-antibody reaction on the surface of a pathological cell or microorganism; an exception for this rule is that IgG 4 cannot activate the classical pathway.
- the present invention relates to the profound change, the coating of CI IA plays in inhibiting not only the CI qrs activation but also the lectin activation.
- the coating of CI IA plays in inhibiting not only the CI qrs activation but also the lectin activation.
- the present invention provides a novel combined immunological-complement activating reaction on cancer cells to promote lysis of these cells, and a test kit that possibly can disclose a possible appearance of cancer in patients, consisting of measurement of the level of CI IA and the complement component "native or naive C4" instead of trying to identify C4 subunits.
- the cancer test diagnostic kit can be used to detect cancer or metastases from cancer at an earlier stage of the cancer disease in patients, who otherwise get identified late, - often up to one year or more into their cancer disease.
- the available methods present now a days to develop and actually produce both diagnostics using the measurement of CI inactivator and naive C4 in blood and may be in spinal fluid, even though it is anticipated that malignant brain tumors - in spite of their more or less exclusive growth in the CNS without metastasizing into the rest of the body, may appear that the CI inactivator released by the malignant brain tumor (primary or secondary), may release the and especially treatment using measurement of the I.V. blood content of CI inactivator and naive C4 in peripheral blood may also reflect tumor growth in the brain.
- mouse monoclonal anti human CI inactivator antibody and mouse monoclonal anti human C4 antibody are used for the production of ELISA kits for the measurement of these components in blood and other body fluids.
- the mouse monoclonal antibody is anticipated to be made by the method described according Proposal for using Creative BioLab's Hybridoma for Monoclonal Antibody manufacturing method for the production of anti human CI inactivator and anti human naive (native) C4 complement component to be used for the testing systems selected, e.g., such as ELISA technology and systems often used at Doctor's office such as Quick Read from Orion
- An in vivo diagnostic approach would be a humanized anti CI IA bound to a tracer such as for instance a radioactive tracer such as Technitium 99, which then could detect for instance via scanning or other measurements producing a picture of where in the body the CI IA is bound, and where there may be metastases.
- a tracer such as for instance a radioactive tracer such as Technitium 99
- This cancer kit can also be used in monitoring cancer patients after surgery - as exemplified in one of the two patients described in this invention, monitor the effect of a cancer treatment whether it is by treating with chemotherapy or an isotope with limited radiation, such as beta rays.
- a humanized monoclonal biphasic anti human CI inactivator antibody labeled to Technicium 99m could be a useful for detection of metastases on images using according to known principles
- novel anti CI IA treatment with repetitive infusions capable of being done with novel human anti CI IA from recombinant human cell lines such as for instance Human Embryonic Kidney cells (HEK) cells and more preferably certain Human HEK cell lines, such as for instance HEK293t or HEK293ts cell lines using a proprietary vector system consisting of pST2 for cloning and pST2-HF3 after cloning which was linearized by Bglll and GcIL restriction enzymes (see figure 3).
- HEK Human Embryonic Kidney cells
- This vector which is a proprietary vector system owned by HumanCell (HumanCell, Naperville, Illinois, USA) and is used to prepare a recombinant human IgG, chosen between any of the subtypes of IgG (IgM antibodies may also be used for this purpose), namely all of those subtype of IgG that can activate the classical pathway of the Complement system, except IgG 4 , which cannot activate the classical pathway of complement.
- the anti CI IA recombinant anti CI IA will carry Fab2 antigen determinants directed against CI IA molecule, and perform binding and activation of the classical pathway for Complement all the way through C9, which will cause "pores" in its binding together with the other cell bound complement component.
- the pore itself is created by a poly-active effect of C9 binding to the cancer cell membrane, thus causing lysis of the cell.
- humanized monoclonal anti human CI IA can be utilized provided it through its binding to the cancer cell can activate the classical pathway of complement.
- the human anti CI IA recombinant IgG or even the humanized monoclonal anti CI IA can be bound or conjugated to a potent chemotherapy, which then can be led all the way directly to the cancer cells carrying CI IA on their plasma membrane, and/or expressing this protein.
- inflammatory protein present as an acute phase protein appearing when patients are infected with microorganisms, such as for instance bacteria.
- a postulated dosage range using specific recombinant human or humanized antihuman CI IA would be starting up with I.V. dosage ranges 1 - 2 gram per kg (such as 1.0 gram per kg, e.g. 1.1 gram per kg, such as 1.2, gram per kg, e.g. 1.3 gram per kg, such as 1.4, gram per kg, e.g. 1.5 gram per kg, such as 1.6, gram per kg, e.g. 1.7 gram per kg, such as 1.8, gram per kg, e.g. 1.9 gram per kg, such as 2.0 gram per kg) given for instance once or twice a month until effect is seen, but depending upon the specificity the doses may be much smaller in size.
- the dosage form comprises 10-50 gram anti CI IA human immunoglobulin, such 11-49 gram anti CI IA human immunoglobulin, e.g. 12-48 gram anti CI IA human immunoglobulin, such 13-47 gram anti CI IA human immunoglobulin, e.g. 14- 46 gram anti CI IA human immunoglobulin, such 15-45 gram anti CI IA human immunoglobulin, e.g. 16-44 gram anti CI IA human immunoglobulin, such 17-43 gram anti CI IA human immunoglobulin, e.g. 18-42 gram anti CI IA human immunoglobulin, such 19-41 gram anti CI IA human immunoglobulin, such 11-49 gram anti CI IA human immunoglobulin, e.g. 12-48 gram anti CI IA human immunoglobulin, such 13-47 gram anti CI IA human immunoglobulin, e.g. 14- 46 gram anti CI
- immunoglobulin e.g. 20-40 gram anti CI IA human immunoglobulin, such 12- 39 gram anti CI IA human immunoglobulin, e.g. 22-38 gram anti CI IA human immunoglobulin, such 23-37 gram anti CI IA human immunoglobulin, e.g. 24-36 gram anti CI IA human immunoglobulin, such 25-35 gram anti CI IA human immunoglobulin, e.g. 26-34 gram anti CI IA human
- immunoglobulin such 27-33 gram anti CI IA human immunoglobulin, e.g. 28- 32 gram anti CI IA human immunoglobulin, such 29-31 gram anti CI IA human immunoglobulin.
- the anti CI IA human immunoglobulin or the recombinant human or humanized antihuman CI IA is administered 1-5 times a month, such as 2-4 times a months, e.g. 2-4 times a month and in an even more preferred embodiment 1 or 2 times a month.
- the anti CI IA human immunoglobulin or the recombinant human or humanized antihuman CI IA is administered weekly or once every second week.
- tumor load is followed by clinical investigation including x-ray, MR Scanning, CT scanning or by using endoscopic methods.
- a preferred administration for is IV administration (intra venous administration).
- the present invention pertains to a method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines.
- CI IA CI esterase inhibitor
- the recombinant CI esterase inhibitor is recombinant human CI esterase inhibitor made in human cells and not in non human (mammal cells such as CHO or alike).
- the HEK cell line is HEK 293T cell line (HumanCell, Naperville, Illinois, USA).
- the human host cell is cultured to a cell density of above 10 3 cells per ml.
- no synthetic protease inhibitor(s) is/are added.
- the said CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
- the amino acid comprises a sequence sharing at least 86 % identity with that set forth in SEQ ID NO. : l or SEQ ID NO. : 2 , such as 20 87 % identity, 88 % identity, 89 % identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, or 99 % identity.
- sequences in ii) and iii) are immunologically
- said CI esterase inhibitor has a carbohydrate moiety, which constitutes from 45-50% of its molecular mass.
- said CI esterase inhibitor comprises sialic acid, such as in the same amounts as native CI esterase inhibitor.
- cells are transfected with IgG to which the gene protope to CI IA antigen of the determinants of an IgG said sequences in ii) and iii) are capable of reacting specifically with an antiserum/polyclonal antibody or a monoclonal antibody raised against a polypeptide having the sequence set forth in i).
- the monoclonal antibody is a mouse monoclonal antibody selected from the group consisting of: Clone 3F4-1D9 (commercially available from Bio-Rad AbD Serotec, Lifespan Biosciences, Merck Millipore, Abnova and United States Biological) , clone LS-C39048 (commercially available form Lifespan Biosciences ) , clone [8D4C12E6 (commercially available from Sino Biological), clone 350507 (commercially available from R&D Systems), clone abxl0017 (commercially available from Abbexa), clone KT28 (commercially available from Thermo Fisher Scientific, Inc.
- Clone 3F4-1D9 commercially available from Bio-Rad AbD Serotec, Lifespan Biosciences, Merck Millipore, Abnova and United States Biological
- clone LS-C39048 commercially available form Lifespan Biosciences
- clones 8F3 and 6C6 commercially available from Abbiotec
- clone 10K343 commercially available from United States Biological
- clone GWB-2F0410 commercially available from Genway
- clone 119-15582 commercially available from Raybiotech
- clone CAB-4608MH commercially available from Creative Biomart
- clone Bll commercially available from Santa Cruz Biotechnology
- clone MAA235Hu22 commercially available from Cloud-Clone Corp
- clones EPR8016, M81, EPR8015, ab54898 commercially available from Abeam).
- the CI esterase inhibitor which comprises an amino acid sequence, which is a subsequence of the sequence set forth in SEQ ID NO. : 1, has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or the sequence set forth in SEQ ID NO. : 2.
- said CI esterase inhibitor comprising a sequence, which has at least 85% identity to the sequences set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 or to a subsequence thereof, has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
- said CI esterase inhibitor consists of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
- iii a sequence which has at least 85% nucleic acid identity with the sequence set forth in i) or ii).
- the nucleic acid comprises a sequence sharing at least 86 % identity with that set forth in SEQ ID NO. : 3 or SEQ ID NO. :4 , such as 87 % identity, 88 % identity, 89 % identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, or 99 % identity.
- said exogenous nucleic acid sequence encoding said CI esterase inhibitor has been inserted into a plasmid vector
- identity is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
- sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
- the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
- the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
- the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
- the two sequences are of different length and gaps are seen as different positions.
- alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm.
- Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990).
- Gapped BLAST may be utilised.
- PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules.
- NBLAST NBLAST
- XBLAST XBLAST
- Gapped BLAST programs
- sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
- sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
- the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment.
- the BLASTN and PSI BLAST default settings may be advantageous.
- the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
- An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation. Production of antibodies
- the invention pertains to a method to the production of monoclonal antibodies against CI IA and native C4
- the invention pertains to a method for producing recombinant human IgG and subtypes of IgG except IgG 4 by transfecting HEK cells using a vector capable of containing the antigenic determinants against CI IA, inducing classical pathway reaction of complement.
- the invention also pertains to the production of recombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI IA developed from the anti CI IA fab.
- the invention also pertains to the production of
- the invention pertains to a human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the placenta, an immortalized cell from the
- the human host cell comprises an exogenous nucleic acid sequence as defined as the Coding region also defined as SEQ ID NO: 3 cloned into pST2 expression vector
- the human host cell expresses a CI esterase inhibitor as defined above.
- the human host cell is 24.
- compositions in another aspect pertains to a recombinant CI esterase inhibitor obtainable by the method above.
- composition comprising a recombinant CI esterase inhibitor, characterised in that
- composition comprises substantially no synthetic protease inhibitors, and/or
- composition is serum free.
- the recombinant CI esterase inhibitor is as defined as above. Use of recombinant CI esterase inhibitor and administration
- the present invention pertains to a recombinant CI esterase inhibitor or a composition comprising recombinant CI esterase inhibitor for use as a medicament.
- the recombinant CI esterase inhibitor or a composition comprising recombinant CI esterase inhibitor may be used for the preparation of a medicament for the treatment and/or prevention and/or reduction of ischemia.
- composition comprising recombinant CI esterase inhibitor may be used for the preparation of a medicament for the treatment and/or prevention and/or reduction of Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation).
- Ischemic stroke reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation.
- composition recombinant CI esterase inhibitor may be used for the
- the recombinant CI esterase inhibitor or a composition recombinant CI esterase inhibitor may be used, for the preparation of a medicament for the treatment of burn wounds (promotion of re- epithelialization, prevention/alleviation of granulation tissue development and scar formation).
- composition recombinant CI esterase inhibitor may be used, for the
- the CI esterase inhibitor or composition may be administered intracerebral ⁇ , intraspinally, intrathecal ⁇ , or intravenously.
- the present invention pertains to a method for preventing, treating and/or alleviating Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor.
- Ischemic stroke reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation
- the present invention pertains to a method for preventing, treating and/or alleviating Inflammation in spinal cord injury comprising administering to a subject in need thereof a recombinant CI esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor.
- the present invention pertains to a method for treating burn wounds (Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor.
- Said CI esterase inhibitor or composition comprising recombinant CI esterase inhibitor may be administered intracerebral ⁇ , intraspinally, intrathecally, or intravenously
- the invention pertains to a an antibody raised against a CI esterase inhibitor according to a method using recombinant human anti human CI inactivator IgG using cloned IgGl cloned into pST2 expression vector
- the antibody is a monoclonal antibody.
- the antibody is a polyclonal antibody/antiserum.
- the antibody may be a polyclonal antibody raised against a composition comprising semi-purified CI esterase inhibitor.
- the said composition may comprise one or more components selected from the group consisting of orosomucoid, alpha2 HS glycoprotein and Zn alpha2 glycoprotein, C-reactive protein.
- the antibody may be conjugated to a detectable marker.
- the marker is a fluorescent marker.
- the marker may be a fluorescein isothiocyanate derivative.
- the chemotherapeutic drug is selected form the group of chemotherapeutic drugs that hit the cancer cells in the M or S phase and not the GO (resting phase), ,cell killing drugs such as Adriamycin, 5 FU.
- the chemotherapeutic drug is be selected form the group consisting of chemotherapy drugs, alkylating agents and platinium drugs.
- the alkylating agents is selected from the group consisting of nitrogen mustards, such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan ® ), ifosfamide, and melphalan, nitrosoureas such as streptozocin, carmustine (BCNU), and lomustine, alkyl sulfonates such as busulfan, triazines such as dacarbazine (DTIC) and temozolomide (Temodar ® ), ethylenimines such as thiotepa and altretamine (hexamethylmelamine).
- nitrogen mustards such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan ® ), ifosfamide, and mel
- the platinum drug is selected from the group consisting of cisplatin, carboplatin and oxalaplatin.
- Antimetabolites interfere with DNA and RNA growth by substituting for the normal building blocks of RNA and DNA. These agents damage cells during the S phase, when the cell's chromosomes are being copied. They are commonly used to treat leukemias, cancers of the breast, ovary, and the intestinal tract, as well as other types of cancer.
- antimetabolites include: ⁇ 5-fluorouracil (5-FU)
- the antibody may be used as a medicament.
- the invention pertains to an antibody raised against a CI esterase inhibitor, wherein the CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
- the antibody may be for use in the manufacture of a medicament for the treatment of a malignant carcinoma, squamous
- carcinomas oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma
- adenocarcinomas such as colon
- pancreas carcinomas of various types and sub types pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney carcinomas bladder carcinomas, malignant brain tumors (e.g.
- the present invention pertains to a method for treating a malignant carcinoma including malignant brain tumors (e.g. glioblastomas or astrocytomas), said method comprising administering to a subject in need thereof an antibody.
- a malignant carcinoma including malignant brain tumors (e.g. glioblastomas or astrocytomas)
- said method comprising administering to a subject in need thereof an antibody.
- the antibody may be used in the manufacture of a medicament for treatment of a malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- the cancer may be a malignant brain tumor such as but not limited to
- One aspect of the present invention provides a method for determining and/or screening cancer in an individual suitable to facilitate the early diagnosis of a cancer. It is another aspect of the present invention to provide a method for monitoring the recurrence of a cancer, status of a cancer or the effect of cancer treatment in an individual. It is a third aspect of the present invention to provide a kit or device for performing the method according to the invention, having a simple and inexpensive design, being quick and easy to use and not requiring the assistance of specialists or the use of specialised equipment.
- the discriminating value is established by measuring the total concentration of CI IA in both a healthy control population and a population with known cancer and thereby determining the discriminating value.
- the discriminating value identifies the cancer population with either a predetermined specificity or a predetermined sensitivity or both, and is based on an analysis of the relation between the concentration values and the known clinical data of the healthy control population and the cancer patient population.
- the discriminating value is established by measuring the total concentration of C4 in both a healthy control population and a population with known cancer and thereby determining the discriminating value.
- the discriminating value identifies the cancer population with either a predetermined specificity or a predetermined sensitivity or both, and is based on an analysis of the relation between the concentration values and the known clinical data of the healthy control population and the cancer patient population.
- the discriminating value determined in this manner is valid for the same experimental set-up in future individual tests.
- the sensitivity of any given diagnostic test define the proportion of individuals with a positive response who are correctly identified or diagnosed by the test, e.g. the sensitivity is 100%, if all individuals with a given condition have a positive test.
- the specificity of a given screening test reflects the proportion of individuals without the condition who are correctly identified or diagnosed by the test, e.g. 100 % specificity is, if all individuals without the condition have a negative test result.
- Sensitivity is defined as the proportion of individuals with a given condition (e.g. cancer), who are correctly identified by the described methods of the invention (e.g. has a positive test-result). Specificity herein is defined as the proportion of individuals without the condition (e.g. cancer), who are correctly identified by the described methods of the invention (e.g. has a negative test result)
- ROC receiver-operating characteristics
- the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease, latent or recent infection versus no infection, or benign versus malignant disease.
- the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results] . This has also been referred to as positivity in the presence of a disease or condition.
- the ROC plot is independent of the prevalence of disease in the sample.
- Each point on the ROC plot represents a sensitivity/- specificity pair corresponding to a particular decision threshold.
- a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false- positive fraction is 0 (perfect specificity).
- the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
- One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
- the specificity of the method according to the present invention may be from 70% to 100%, more preferably 80% to 100%, more preferably 90% to 100%.
- the specificity of the invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 20 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
- the sensitivity of the method according to the present invention may be from 70% to 100%, more preferably 80% to 100%, more preferably 90% to 100%.
- the sensitivity of the method according to the present invention may be from 70% to 100%, more preferably 80% to 100%, more preferably 90% to 100%.
- 25 invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
- the levels of CI IA and native C4 are compared to a set of reference data or a reference value such as the cut-off value to determine whether the subject is 30 at an increased risk or likelihood of e.g. cancer. To determine whether the patient is at increased risk of developing e.g.
- cut-off must be established. This cut-off may be established by the laboratory, the physician or on a case by case basis by each patient. Alternatively cut point can be determined as the mean, median or geometric mean of the negative control group ((e.g. healthy population, healthy individual, population with known cancer or individual with known cancer) +/- one or more standard deviations or a value derived from the standard deviation)
- the present inventors have successfully identified a new marker for measuring a cell-mediated response to an antigen.
- the concentration of the marker IP-10 is increased in subjects with a cell mediated immune-response to an antigen.
- IP-10 appears to be an efficient marker for detection of e.g. infection with M. tuberculosis.
- the sensitivity is higher than any other established marker and the specificity is comparable or better than with any other established marker, see e.g.
- Statistical reasoning can for example be based on the risk of having the disease depending on age, occupation, exposure, genetic background, HLA- type.
- Cut-off points can vary based on specific conditions of the individual tested such as but not limited to the risk of having the disease, occupation, geographic residence or exposure. Cut-off points can vary based on specific conditions of the individual tested such as but not limited to age, sex, genetic background (i.e. HLA-type), acquired or inherited compromised immune function (e.g. HIV infection, diabetes, patients with renal or liver failure, patients in treatment with immune-modifying drugs such as but not limited to corticosteroids,
- TNF-a blockers TNF-a blockers, mitosis inhibitors.
- methods for screening/monitoring/determining cancer are processes of decision making by comparison.
- reference-values based on subjects having the disease and/or subjects not having the disease, infection, or condition of interest are needed.
- the cut-off level (or the cut-off point) can be based on several criteria including the number of subjects who would go on for further invasive diagnostic testing, the average risk of having and/or developing e.g. cancer to all the subjects who go on for further diagnostic testing, a decision that any subject whose patient specific risk is greater than a certain risk level should go on for further invasive diagnostic testing or other criteria known to those skilled in the art.
- the cut-off level can be adjusted based on several criteria such as but not restricted to certain group of individuals tested. E.g. the cut-off level could be set lower in individuals with known cancer, cut-off may be higher in groups of otherwise healthy individuals with low risk of developing cancer.
- the present invention discloses a method for determining if a subject is likely of having cancer, which comprises:
- one aspect of the present invention relates to a method of diagnosing cancer, comprising the steps of a) incubating a sample obtained from an individual with anti-Cl IA and incubating another sample obtained from the same individual with anti-
- the discriminating value is a value which has been determined by measuring the parameter or parameters in both a healthy control population and a population with known cancer thereby determining the discriminating value which identifies the cancer population with either a predetermined specificity or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control population and the cancer patient population, such as it is apparent from the detailed discussion in the examples herein.
- the discriminating value is a value which has been determined by measuring the parameter or parameters in both a healthy control population and a population with known cancer thereby determining the discriminating value which identifies the cancer population with either a predetermined specificity or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control population and the cancer patient population, such as it is apparent from the detailed discussion in the examples herein.
- the level threshold of CI IA useful as a cut-off value was found to be in the range of but not limited to 40 mg/100 ml sample. Preferably the cut-off is in the range between 15 - 40 mg/100 ml.
- Normal serum concentrations of CI IA is in the range from 15-40 mg/ml serum whereas normal serum concentrations of native C4 is in the range from 15-50 mg/lOOml.
- the cut-off value indicating a suspected level of CI IA would be 45 mg/100 ml (and a level of 60 mg/100 ml of native C4, see below).
- the level threshold of native C4 useful as a cut-off value was found to be in the range of but not limited to 20 mg/100 ml to 50 mg/100 ml.
- the cut-off is in the range between 20 mg/100 ml and 60 mg/100 ml
- the cut-off value is over 60 mg/100 ml
- the multivariate DISCRIMINANT analysis and other risk assessments can be performed on the commercially available computer program statistical package Statistical Analysis System (manufactured and sold by SAS Institute Inc.) or by other methods of multivariate statistical analysis or other statistical software packages or screening software known to those skilled in the art.
- Statistical Analysis System manufactured and sold by SAS Institute Inc.
- changing the risk cut-off level of a positive test or using different a priori risks which may apply to different subgroups in the population could change the results of the discriminant analysis for each patient.
- the amount of CI IA and native C4 may be measured by a device, said device is selected from the group consisting of an assay, an immunoassay, a stick, a dry-stick, an electrical device, an electrode, a reader (spectrophotometric readers, IR- readers, isotopic readers and similar readers), histochemistry, and similar means incorporating a reference, filter paper, colour reaction visible by the naked eye.
- the method according to the invention can equally well be used for monitoring the response to treatment and the progress of the cancer as rising CI IA values in combination with decreasing C4 values may suggest that a patient could have a negative development of the cancer. In such cases the
- the cancer may be selected from the group consisting of... malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
- adenocarcinomas such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney carcinomas bladder carcinomas, ; including malignant brain tumors (e.g. glioblastomas or astrocytomas).
- glioblastomas grade III-IV, gliomas, glioblastoma multiforme
- oligodendrogliomas ependymoma, medulloblastoma.
- the method according to the invention may be used both for an individual and for an entire population, but more appropriately to a population already identified as having an increased risk of developing cancer, e.g. individuals with a genetic disposition, individuals who have been exposed to carcinogenic substances, or individuals with cancer-predisposing non-malignant diseases.
- the individual When an individual has been identified as having high CI IA levels in combination with low C4 in his or her excreta, the individual should be referred for further examination. If a cancer is found, the patient could be offered surgery, radiation or adjuvant anti-neoplastic therapy aiming at curing the patient of cancer.
- CI IA and native C4 may be used for predicting the prognosis of subjects diagnosed with cancer.
- the method according to the present invention may help to predict the course and probable outcome of the cancer, thus assisting the skilled artisan in selecting the appropriate treatment method and predict the effect of a certain treatment for the condition.
- CI IA and native C4 may be used for monitoring subjects diagnosed with cancer.
- the method according to the present invention may help to assess efficacy of treatment during and after termination of treatment e.g. monitoring and predicting possible recurrence of the cancer.
- malignant carcinoma squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney
- carcinomas bladder carcinomas including malignant brain tumors (e.g. glioblastomas or astrocytomas).
- malignant brain tumors e.g. glioblastomas or astrocytomas.
- the method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of cancer by measuring the level of CI IA and native C4 according to the invention and correlating the levels measured to a pre-specified levels, indicating the presence or absence of cancer.
- CI IA and native C4 or other biological markers are measured by conventional analytical methods, such as immunological methods known to the art.
- Measurements of biological markers such as CI IA and C4 can be combined with measurements of other molecules at gene, RNA, or protein level in accordance with the teachings herein.
- detection of biological markers such as CI IA and C4 may be made at the protein or nucleic acid levels. Consequently, reference to the presence or level of said CI IA, C4 and other relevant biological markers includes direct and indirect data.
- high levels of CI IA and native C4 mRNA are indirect data showing increased levels of CI IA and C4 respectively.
- Ligands to CI IA and C4 are particularly useful in detecting and/or quantitating these molecules.
- Antibodies to biological markers such as CI IA and native C4 are particularly useful. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays, xMAP multiplexing, Luminex, ELISA and ELISpot. Reference to antibodies includes parts of antibodies, "mammalianized” (e.g. humanized) antibodies, polyclonal, recombinant or synthetic antibodies and hybrid and single chain antibodies.
- Both polyclonal and monoclonal antibodies are obtainable by immunization with the biological marker to be measured e.g. CI IA and native C4 or antigenic fragments thereof and either type is utilizable for immunoassays.
- the biological marker to be measured e.g. CI IA and native C4 or antigenic fragments thereof and either type is utilizable for immunoassays.
- the methods of obtaining both types of sera are well known in the art.
- Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the biological marker (e.g. CI IA and C4), or antigenic part thereof, collecting serum or plasma from the animal and isolating specific sera by any of the known immuno-adsorbent techniques.
- the biological marker e.g. CI IA and C4
- antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
- the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
- Detection can also be obtained by either direct measure of the biological marker (e.g. CI IA and C4) using specific antibody in a competitive
- CFIPA fluorescent polarization immunoassay
- DIFP dimerization induced fluorescence polarization
- the presence or level of immune effecter may be determined by a Sandwich ELISA method, where the Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form, or variation of that method most recently described for the use of measuring complement components (Kotimaa JP et al. J Immunol Methods. 2015 Apr;419 : 25-34).
- Luminex beads method from Bio- Rad called Luminex xMAP technology
- Bio-Plex Pro TM magnetic multiplex assays provide researchers the most relevant multiplexing solutions.
- Luminex ELISPOT
- mRNA based techniques like RT-PCR or Intracellular flow cytometri.
- ELISPOT In order to idenetify patients, who could be used as donors to identify human anti CI inactivator, ELISPOT, could be a convenient method as a screening method for screening Quincke edema, lymphoproliferative or lymphoma patients for autoimmune antibodies against CI inactivator, ELISPOT would be a suitable method, as it was developed by Cecil Czerkinsky's group in
- Sandwich ELISA method could be convenient method for testing the level of CI inactivator and native C4, as described by Kotimaa et al used on complement components as such (Kotimaa JP et al. J Immunol Methods. 2015 Apr;419 : 25-34) because the test system only have to be active in the mg area, and more difficult to be used in other areas where one is looking for microgram amount.
- the sample is derived from the group consisting of blood, serum or spinal fluid (if brain cancer is suspected)
- the sample is derived from blood.
- the blood collection tube is treated with anticoagulant (e.g. heparin, or ACD, etc).
- anticoagulant e.g. heparin, or ACD, etc.
- whole blood is the preferred and most convenient sample
- the present invention extends to other blood derived sample such as pleural or ascites fluids, and spinal fluid sample.
- the present invention thus relates to a method, wherein the sample is derived from blood, urine, pleural fluid, bronchial fluid, oral washings, tissue biopsies, ascites liquid, pus, cerebrospinal fluid, aspirate, and/or follicular fluid.
- blood is maintained in the presence of an anticoagulant (preferably heparin, alternatively e.g. citrate or EDTA).
- an anticoagulant preferably heparin, alternatively e.g. citrate or EDTA.
- the anticoagulant is present in the blood collection tube when blood is added.
- the use of blood collection tubes is preferably but not necessarily compatible with standard automated laboratory systems and these are amenable to analysis in large-scale and random access sampling. Blood collection tubes also minimize handling costs and reduce laboratory exposure to whole blood and plasma and, hence, reduce the risk of laboratory personnel from contracting a pathogenic agent such as but not limited to human immunodeficiency virus.
- Alliquots of whole blood may be in volumes ranging from 10 ⁇ -4000 ⁇ , such as but not limited to 50 ⁇ _, 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , 800 ⁇ , 900 ⁇ , 1000 ⁇ , 1100 ⁇ , 1200 ⁇ , 1300 ⁇ , 1400 ⁇ , 1500 ⁇ , 1600 ⁇ , 1700 ⁇ , 1800 ⁇ , 1900 ⁇ , 2000 ⁇ , 2100 ⁇ , 2200 ⁇ , 2300 ⁇ , 2400 ⁇ , 2500 ⁇ , 2600 ⁇ , 2700 ⁇ , 2800 ⁇ , 2900 ⁇ or 3000 ⁇ . Kit
- the invention further relates to a kit for measurement of the concentration of CI IA and native C4 in excreta .
- kit may consist of a dipstick for determining CI IA and native C4 in excreta however, other options may be but is not limited to Activity Assay (such as zymography), immunologic assays or a Colour Reaction kit.
- the present invention further contemplates a kit for performing the method of the present invention.
- the kit is conveniently in compartmental form with one or more compartments adapted to receive a sample from a subject such as whole blood, serum, purified cells, biopsies or other material . That
- compartment or another compartment may also be adapted to contain heparin where the sample is whole blood .
- the kit is in a form which is packaged for sale with a set of instructions.
- the instructions would generally be in the form of a method of the present invention - i.e. for diagnosing cancer in a subject.
- the kit contains antigen and monoclonal or polyclonal antibodies against Cl-IA and C4, which is specifically reacting with CI IA and C4 respectivly in an immune-assay, or specific binding fragments of said antibodies for use as a diagnostic reagent.
- the contemplated kit of the present invention may be in a multicomponent form wherein a first component comprises a multiplicity of blood collection tubes, a second component comprises an antibody-based detection for CI IA, third component comprises an antibody-based detection means for C4, a fourth component comprises a set of instructions.
- the assay may also be automated or semi-automated and the automated aspects may be controlled by computer software.
- the assay of the present invention may be automated or semi-automated for high throughput screening or for screening for a number of immune effecters from the one subject.
- the automation is conveniently controlled by computer software.
- the present invention contemplates a computer program product, therefore, for assessing the presence or absence or the level of CI IA and C4, said product comprises:
- code that receives, as input values, the identity of a reporter molecule associated with a labelled antibody or mRNA
- code that compares said input values with reference values to determine the level of reporter molecules and/or the identity of the molecule to which the reporter molecule is attached
- Still another aspect of the present invention extends to a computer for assessing the presence or absence or level of CI IA and C4, said computer comprises: (1) a machine-readable data storage medium composing a data storage material encoded with machine- readable data, wherein said machine- readable data I comprise input values which identify a reporter molecule associated with a labelled antibody or mRNA; (2) a working memory for storing instructions for processing said machine- readable data, (3) a central-processing unit coupled to said working memory and to said machine readable data storage medium, for processing said machine readable data to compare said values to provide an
- the invention pertains to a method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
- CI IA inactivator
- C4 complement component native C4
- the first parameter is the total concentration of CI IA.
- the second parameter is the total concentration of native C4.
- the second parameter is the combination of the concentration of total native C4
- the at least one excreta is selected from the group consisting of blood, serum, saliva, spinal fluid, cerebro spinal fluid, pleura fluid, ascites fluid and urine.
- the concentration of CI IA and/or the concentration of native C4 is obtained any time before operation.
- the concentration of CI IA and/or the concentration of native C4 is obtained any time after an operation such as 2 weeks post- operation, 1 month post-operation, 1.5 month post-operation, 2 months post- operation, 3 month post-operation, 4 months post-operation, 5 month post- operation, 6 months post-operation, 7 month post-operation, 8 months post operation.
- the combination is performed by logistic regression analysis.
- the first discrimination value is determined by determining the total concentration of CI IA in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
- the second discrimination value is determined by determining the total concentration of C4 in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
- the determination of the concentration is performed by means of an immunoassay or an active assay.
- the immunoassay is an ELISA.
- the active assay is zymography.
- the cancer is selected from the group consisting of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
- adenocarcinomas such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar
- carcinomas breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- the method may be used for detection of early stage cancer or for detection of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
- adenocarcinomas such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar
- carcinomas breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- the method may also be used for monitoring the response to cancer
- the method may also be used for monitoring the recurrence of a cancer.
- the invention pertains to a kit for performing the method above, wherein said kit comprises CI IA and native C4.
- the invention pertains to a method of diagnosing rejection of transplants, said method comprising the steps of a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
- the invention pertains to a method of diagnosing cancer, said method comprising the steps of
- Figure 1 discloses the concentration of CI IA and complement component C4 in blood is visualized in three rows for three categories of patients, measured using Laurell Immunoelectrophoresis.
- Normal serum concentration range of CI IA is 15 - 40 mg /100 ml.
- Normal serum concentration range for C4 is 15 - 50 mg/ 100 ml.
- native C4 in blood was as low as 4 mg/100 ml Healthy subjects consisting of blood donors and patients suffering from non- malignant disease had values for both CI IA and C4 in the same range, whereas cancer patients had in major cases higher concentrations of both CI IA and native C4.
- Figure 2 discloses a simplified drawing of the complement system, targeting the classical pathway of a complement reaction, which is caused by an antigen-antibody reaction, and during chain of reactions occurring through the pathway until C9, the individual complement components are split up after activation to be bound to the pathogen, e.g., the cell, to perform opsonisation, meaning enhancing phagocytosis of antigen, chemotaxis which is attracting macrophages and neutrophils, ending up after sequential binding of part of the split products of C4 to C9, where C9 consists of several fractions that results in a binding of part of the C4, C2, C3, C5 up to C9, catalyzing the enzymatic degradation of covalently bound complement proteins actually ending up with a perforation of the plasma membrane described as "pores" in the plasma membrane by multiple part of the enzymatic molecule C9, resulting in cell lysis or cell death, in many eventually ending up with agglutination and further phagocytosis.
- the pathogen
- Figure 3 discloses the pST2 vector for cloning.
- pST2-HF3 after cloning.
- pST2- HF3 vector was linearized by Bglll and Pcil restrictions enzymes.
- a HEK cell line adapted to a suspension cell culture expanded in serum-free medium.
- the cDNA coding region of Clinactivator is cloned into pST2 expression vector (HumanCell Co. Proprietary) at Srfl site to contruct pST-HF3.
- pST2-HF3 construction CI inactivator coding region will be sequenced and confirmed.
- the cell line is transfected.
- the cells are then selected and cells appearing to be good producers will be pooled and transferred, plated and after ⁇ 7 days, and adapted as described to suspension in serum-free medium.
- the cells are then grown in shaker flasks in a 37°C C0 2 humidified incubator.
- the cell morphology of the suspension culture is then compared to the monolayer culture. Supernatant is tested for the presence of the protein targeted, - in this case the CI inactivator.
- the same system is used when producing recombinant IgGi , e.g., after the study and evaluation of the paratope for the antigenic determinant (e.g., human CI inactivator), and have been annealed to the antigen determinant in the F(ab') 2 "Y" end of the recombinant immunoglobulin.
- the antigenic determinant e.g., human CI inactivator
- Figure 4 discloses the activity of CI inhibitor (C1IA) on carcinoma cells and non-malignant cells in culture using Leitz Cytophotometric measurement of cells on a Leitz MPVlCytophotometer mounted on an Orthoplan phase contrast microscope.
- CI inhibitor C1IA
- FITC Fluorescein Isothiocyanate
- anti C1IA anti C1IA antibody
- phase contrast microscope one cell was centered into the measuring diaphragm of the Cytophotometer and the excitatation was measured for FITC activity. A number of 100 cells from each cell culture was counted.
- First column shows the distribution of percentage of CI IA "positive" primary carcinoma cell cultures, - showing specific binding of FITC conjugated rabbit anti human CI IA as evidenced by excitation measurements done from the first explantation of cells on micro-slides.
- the percentage-range of CI IA positive carcinoma cells in the cell cultures measured was plotted into a diagram and ranged from approximately 25% to >75%.
- Second column shows the distribution of carcinoma cell cultures sub-cultured in Falcon flasks and re-explanted on micro-slides in Leighton tubes and incubated in a humidified 37°C C0 2 incubator. These measurements showed approximately the same distribution in percentage of CI IA positive cells.
- the third column show carcinoma cell cultures pre-incubated with unconjugated polyclonal rabbit anti CI IA, then washed times 3 in Phosphate buffered saline, (PBS buffer) at pH7.2-7.4 and re-incubated with FITC-conjugated polyclonal rabbit anti human CI IA. No cells showed any specific excitation.
- the fourth column incubated with FITC conjugated rabbit anti human CI IA and measured as described above showed no specific excitation when measured with the above described
- Cytophotometer indicating no specific binding of anti CI IA on these non- malignant cells, which included among others human mesenchymal cells and fibroblast cultures.
- Figure 5 discloses a 28 month old infant (patient No 1) who was treated with intravenous infusion of 180 ml (10% porcine semipurified IgG) of xenogeneic anti CI IA treatment ClIA prepared from pigs immunized with semipurified CI IA; CI IA and native C4 levels was monitored through the observation period where the infant was treated with chemotherapy and anti CI IA infusion.
- Figure 6 discloses patient No. 1. Blood count of B lymphocyte changes in a 28 months old child with reticulosarcoma. Notice the significant increase of monocytes (MONO) immediately in connection with xenogeneic semipurified anti CI IA infusion, and Vincristine followed by a relatively high count of Monocytes followed by a sharp decrease of monocytes after the last cycle of Vincristine,xxxx On figure 4, it can be compared to the steady state of CI IA at the level of around 60+ mg/100 ml, but a constant increase of native C4. Immediately in connection with the infusion of the anti CI IA, a sharp increase of IgG lymphocytes are noted, which afterwards falls sharply during the Vincristine treatment. The increase in serum IgM after 9-14 days after the infusion of xenogeneiceic (porcine) semipurified Anti CI IA, indicating a possible active immunization against most probably porcine induced proteins.
- MONO monocytes
- Figure 7 discloses a 53 year old female with a disseminated mammary carcinoma with metastases to bone and to the resection zone on the skin, with several cutaneous metastases en cuirass.
- the patient started out with a CI IA in the blood of 80+ mg/100 ml and a native C4 at ⁇ 58 mg/100 ml.
- the patient showed an initial minor increase in both CI IA and native C4, where CI inactivator within 1 month was around 55 mg/100 ml while her cutaneous metastases started to flatten.
- Figure 8 discloses the first (1.) increase of IgM as a most probable response on the first Xenogeneic (porcine) anti CI IA peaking after approx.. 2 weeks, and the second xenogeneic (sheep) anti CI IA peaking a few days to approximately 14 days after the second xenogeneic anti CI IA, showing what also is postulated in
- Figure 9a exemplifies an antigen -antibody reaction on the surface of a foreign cell or microorganism, followed by activation of the complement components, starting with binding Clqrs to the immune complex caused by the binding of the antibody to the antigen (e.g., of type IgGl, igG2, IgG3 or IgM).
- the antigen e.g., of type IgGl, igG2, IgG3 or IgM.
- the Clqrs are split up into units and has activated and C4 whereof parts is bound to the surface of the "target” cell and split into other subunits, parts of C2 and parts of the complement components following the activation of C2 down to C9 is bound to the surface of the "target” cell.
- the multi components of C9 penetrates the plasma membrane of the cell or microorganism, and extracellular fluid equilibrates with the internal of the cell, which then go into lysis.
- proteins or protein fragments constitute the complement system, including serum proteins, membrane-like proteins, and cell membrane receptors. These proteins constitute 5% of the globulin fraction of blood serum and they are also serving as opsonins (an antibody or other substance that binds to foreign microorganisms or cells, making them more susceptible to phagocytosis).
- Figure 9b discloses the CI Inactivator (CI IA) coat on the cancer cell membrane (the plasma cell membrane) is enlarged in this drawing in order for the reader to note the presence of the CI inactivator coat, and its inhibition and blocking of the activation of the Clqrs complex, which now cannot take place, whereby complement C4 will not be activated, which then results in no activation of C2 which means that the remaining classical complement activation ending with C9 will not take place. Therefore no Immune reaction, which includes the complement cascade, will take place on the Cancer cells coated with CI inactivator (C1IA). Antigen-Antibody cannot activate Complement CI for it to bind and activate complement component C4, (C4 is blocked). Therefore, no Complement Cascade will take place. The Result: no cancer cell lysis, no cancer cell death.
- CI IA CI Inactivator
- Figure 9c discloses the CI IA antigen coating the cancer cell is now exposed to the injected exogenous emonoclonal recombinant anti CI IA antibody (e.g., IgGi) in an optimal amount and titre will neutralize the CI IA coat on the cancer cells, so that the CI IA on the cancer cell cannot inhibit the activation of the complement component Clqrs complex, which again will bind and activate complement component C4, which then will activate the remaining part of the complement system from C2 - C9 without interference from the now neutralized CI IA on the coat of the cancer cell.
- the CI IA antibody e.g., IgGi
- the antigen-antibody immune reaction Ag(Cl IA)-Ab(IgGl) is now able to activate the Complement Clqrs which now can activate C4 to C4b, and activate the C2 C9 Cascade. Result: Cancer Cell Lysis or Cell Death !
- CI inactivator an exogenous mechanism is used to activate an endogenous, in vivo protein (CI inactivator), which is an induced antigen antibody effect to inactivate the CI inactivator and at the same time utilize the in vivo originated antigen reaction with the infused external antibody (e.g., external recombinant IgG) directed against CI inactivator, the binding of which will now bind Clqrs complex which will activate the C4 complement component.
- CI inactivator an endogenous, in vivo protein
- Figure 9c is a theoretical schematic drawing showing how an injected exogenous humanized monoclonal recombinant antibody (e.g., IgGi) with its antigenic determinants will firmly bind to the CI IA coating the cancer cells.
- IgGi humanized monoclonal recombinant antibody
- the protein itself meaning the humanized recombinant anti CI IA monoclonal recombinant immunoglobulin which by binding to the antigen (CI IA) on the surface of the cancer cell now can activate the complement all the way from binding of the complement component CI qrs complex because of the lack of inhibitory effect from the neutralized CI IA coat on the cancer cell, because at the same time as this IgG such as for instance IgGi and other IgG molecules (minus IgG 4 ) works as an antibody with determinants against CI IA.
- the CI IA especially coating the cancer cells is neutralized by the anti CI IA antibody (e.g., IgGl) and cannot hinder the activation of the Clqrs, which again activate the C4, which in cancer patients are found to be abundantly in place in the blood circulation due to the previous blockage of C4 activation because of the effect of the previous active, but now neutralized CI IA, especially on the cancer cells' coat or cell membrane also called the plasma membrane.
- the anti CI IA antibody e.g., IgGl
- SEQ ID NO: 1 Plasma protease CI inhibitor precursor [Homo sapiens] . Signal peptide: aa. 1-22; mature peptide. Aa. 23-500 (NCBI Reference Sequence : NP_000053.2)
- SEQ ID NO: 2 Plasma protease CI inhibitor precursor [Homo sapiens] .
- SEQ ID NO: 3 Signal peptide - homo sapiens serpin peptidase inhibitor, clade G (CI inhibitor).
- SEQ ID NO: 4 Homo sapiens serpin peptidase inhibitor, clade G (CI inhibitor).
- HEK cell line is HEK 293T cell line (HumanCell, Naperville, Illinois, USA)
- said CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
- CI esterase inhibitor comprises sialic acid, such as in the same amounts as native CI esterase inhibitor.
- cells transfected with IgG to which the gene protope to CI IA antigen of the determinants of an IgG said sequences in ii) and iii) are capable of reacting specifically with an antiserum/polyclonal antibody or a monoclonal antibody raised against a polypeptide having the sequence set forth in i).
- the monoclonal antibody is a mouse monoclonal antibody selected from the group consisting of: Clone 3F4- 1D9 (commercially available from Bio-Rad AbD Serotec, Lifespan Biosciences, Merck Millipore, Abnova and United States Biological) , clone LS-C39048 (commercially available form Lifespan Biosciences ) , clone [8D4C12E6 (commercially available from Sino Biological), clone 350507 (commercially available from R&D Systems), clone abxl0017 (commercially available from Abbexa), clone KT28 (commercially available from Thermo Fisher Scientific, Inc. and Abbiotec), clones 8F3 and 6C6 (commercially available from
- : 1 has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or the sequence set forth in SEQ ID NO. : 2.
- subsequence thereof has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
- AAME hydrochloride
- exogenous nucleic acid sequence comprises a sequence selected from the group consisting of
- iii a sequence which has at least 85% nucleic acid identity with the sequence set forth in i) or ii).
- a human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the human cells such as HEK cells, PerC6 cells, amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a CI esterase inhibitor.
- a recombinant CI esterase inhibitor obtainable by the method according to any one of claims 1-19.
- COMPOSITION 26 A composition comprising a recombinant CI esterase inhibitor
- composition comprises substantially no synthetic protease inhibitors, and/or
- composition is serum free.
- composition according to claim 25, wherein said recombinant CI esterase inhibitor is as defined in claim 24.
- composition according to claim 25 or 26, for use as a medicament for use as a medicament.
- a method for preventing, treating and/or alleviating Inflammation in spinal cord injury comprising administering to a subject in need thereof a
- composition according to claim 25 or 26 composition according to claim 25 or 26.
- a method for treating burn wounds, Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor according to any of claim 20, or a composition according to claim 25 or 26.
- composition comprises one or more components selected from the group consisting of orosomucoid, alpha2 HS glycoprotein and Zn alpha2 glycoprotein, C-reactive protein.
- composition comprises one or more components selected from the group consisting of orosomucoid, alpha2 HS glycoprotein and Zn alpha2 glycoprotein, C-reactive protein.
- chemotherapeutic drug is selected form the group consisting of chemotherapy drugs, alkylating agents and platinium drugs.
- alkylating agents is selected from the group consisting of nitrogen mustards, such as
- mechlorethamine nitrogen mustard
- chlorambucil chlorambucil
- cyclophosphamide Cytoxan ®
- ifosfamide ifosfamide
- melphalan nitrosoureas
- streptozocin carmustine
- BCNU carmustine
- lomustine alkyl sulfonates
- triazines such as dacarbazine (DTIC) and temozolomide (Temodar ® )
- ethylenimines such as thiotepa and altretamine (hexamethylmelamine).
- an antibody raised against a CI esterase inhibitor wherein the CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
- a malignant carcinoma squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney carcinomas bladder carcinomas, malignant brain tumors (e.g.
- glioblastomas or astrocytomas astrocytoma
- glioblastomas grade III-IV astrocytoma
- gliomas glioblastoma grade III-IV
- gliomas glioblastoma multiforme
- oligodendrogliomas ependymoma
- medulloblastoma astrocytoma
- glioblastomas grade III-IV gliomas
- glioblastoma multiforme oligodendrogliomas
- ependymoma medulloblastoma
- adenocarcinomas such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- a malignant brain tumor e.g. glioblastoma or an astrocytoma.
- a method for detecting and/or screening and/or monitoring cancer in an individual comprising determining :
- CI IA inactivator
- CI IA inactivator
- second parameter represented by the concentration of complement component native C4 in at least one excreta from the individual wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
- the at least one excreta is selected from the group consisting of blood, serum, saliva, spinal fluid, cerebro spinal fluid, pleura fluid, ascites fluid and urine.
- concentration of CI IA and/or the concentration of native C4 is obtained any time before operation.
- concentration of CI IA and/or the concentration of native C4 is obtained any time after an operation such as 2 weeks post-operation, 1 month post- operation, 1.5 month post-operation, 2 months post-operation, 3 month post- operation, 4 months post-operation, 5 month post-operation, 6 months post- operation, 7 month post-operation, 8 months post operation.
- the first discrimination value is determined by determining the total concentration of CI IA in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
- the second discrimination value is determined by determining the total concentration of C4 in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
- determination of the concentration is performed by means of an immunoassay or an active assay.
- immunoassay is an ELISA. 70. A method according to any of the preceding claims, wherein the active assay is zymography.
- cancer is selected from the group consisting of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon
- pancreas carcinomas of various types and sub types pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- adenocarcinomas such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
- kits for performing the method according to claim 57-71 wherein said kit comprises CI IA and native C4.
- a method of diagnosing rejection of transplants comprising the steps of
- a method of diagnosing cancer comprising the steps of a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample, b) incubating a blood sample or exsudate or transudate in connection with a suspected rejection of (transplanted) tissue from a human with anti-C4d and determining the C4d level in said sample, c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4d level with a second reference-level , e) determining whether said human is likely of experience a tissue or bone marrow rejection if the level of Cl-IA is normal or lower than 15 mg/100 ml and if the level of C4d is higher than 50 mg/100 ml.
- First category of individuals were "healthy subjects" from a Copenhagen based blood bank.
- Second category was blood samples from patients suffering from benign (non cancerous or malignant diseases), and the third category was blood samples from patients suffering from cancer.
- the CI inactivator - and the naive C4 level found in cancer patients indicate that by measuring the I.V.
- the very inexpensive test might be proven valuable when treating cancer patients with cytostatics or with monoclonal antibodies.
- Figure 2 shows a simple overview of the classical pathway of the complement system with emphasis on the CI inhibitor or CI inactivator (CI IA) controls and inhibit the activity at the level of Clqrs complex in the classical pathway and in the lectin pathway.
- CI IA CI inhibitor or CI inactivator
- dissociation of the Clqrs complex is inhibited from splitting up into Clq, Clr and Cls, and thereby the complement component C4 will be decreasingly dissociated to the opsonin part of the C4 and the C4, which is bound to the target in the case of a microorganism or a cell, part of the C4 should be bound directly to the cell surface and a part, called C4b, and a relatively C4a, a weak chemo attractant, is released.
- Figure 2 is a simplified drawing of the complement system, targeting the classical pathway of a complement reaction, which is caused by an antigen- antibody reaction, and during chain of reactions occurring through the pathway until C9, the individual complement components are split up after activation to be bound to the pathogen, e.g., the cell, to perform opsonisation, meaning enhancing phagocytosis of antigen, chemotaxis which is attracting macrophages and neutrophils, ending up after sequential binding of part of the split products of C4 to C9, where C9 consists of several fractions that results in a binding of part of the C4, C2, C3, C5 up to C9, catalyzing the enzymatic degradation of covalently bound complement proteins actually ending up by creating so called "pores" in the plasma membrane multiple part of the enzymatic molecule C9, resulting in cell lysis or cell death, in many cases eventually ending up with agglutination and further phagocytosis.
- the pathogen e.g., the cell
- the "classical pathway” of complement involved in antigen-antibody reaction on cells is the pathway that has been blocked or inhibited on certain carcinoma cells due to the coating with CI IA of many of these cells hindering a disassociation of the Clqrs complex that again block the binding of the C4 molecule to the cell surface as well as the development of C4b, C4c and possibly C4d subunits.
- the primary malignant tumor that most commonly metastasizes to the skin in women is breast cancer, which can be manifested through papulonodular lesions, erysipeloid or sclerodermiform infiltration, en cuirasse.
- Samples from malignant tissue were obtained during operation, transferred to sterile tubes containing Hanck's Stock solution, and kept at 4°C, brought down to the research laboratory, transportation time, did not exceed 3 hours.
- the cells were then cut from small biopsies, washed trypsinized and washed in Eagle MEM, centrifuged and explanted to obtain a monolayer on sterile micro- slides in Eagle MEM with fetal bovine serum in Leighton tubes for testing after 3 to 5 days post culturing, whereafter the cells on the microslides were tested for the presence of CI IA.
- the cells from the same patients were also explanted in medium into 25 cc sterile Falcon flasks for monolayer culturing for longer periods. Control cells from benign tissue were obtained from patients with no overt cancer during surgery and processed as above. The cells cultured in Falcon flasks were later on also used for CI IA after some weeks, where a number of cells were removed by trypsination and explanted on new micro-slides for second or third testing for C1IA
- Non-conjugated rabbit anti CI IA were used for control experiments.
- Excitation light source was an HBO 200 lamp house.
- the Excitation light passed through a 1mm BG12 filter and a narrow band filter KP490 and a K530 barrier filter excluding non-specific signals. Borderline measurements between specific and non-specific fluorescence is described by Osther and Dybkjaer (Osther K, Dybkjaer E, Complement components on the surface of normal lymphocytes. Scand. J. Haematol., 1974, 13 : 24-32). Control experiments using incubation of the cells with non-conjugated rabbit anti human CI IA, followed by consecutive washings in PBS buffer at pH 7.2- 7.4, followed by second incubation with FITC conjugated rabbit anti Human CI IA. The fluorescence measurement was plotted onto diagrams.
- Figure 4 shows the primary carcinoma cell cultures measured after being incubated for 30 minutes at 37 degrees C, rinsed and measured in the Leitz microscop+e. The results of the measurement of several carcinoma cultures were plotted in and the percentage of cells showing FITC activity was plotted in to a table as shown in figure 4, several of the primary cell cultures sub- cultured from each primary carcinoma culture was explanted on micro-slides, incubated with FITC polyclonal anti CI esterase inhibitor (Behring Institute) and measured as described above.
- the percentage of cells showing FITC activity were plotted in as a percentage of 100 cells.
- rabbit polyclonal anti human CI IA FITC antibody from Behring Institute
- CI IA unconjugated rabbit polyclonal anti CI esterase inhibitor also called CI IA (from Behring Institute)
- CI IA unconjugated rabbit polyclonal anti CI esterase inhibitor
- CI inhibitor ClIA
- Leitz MPV 1 Cytophotometer that could measure cells and show that the cells bound polyclonal rabbit anti CI IA - FITC (from Behring Institute, Marburg, Germany).
- a number of 100 cells from each cell culture was counted and FITC excitation was measured.
- the results were expressed in percentage of cells showing FITC conjugated anti CI IA binding,
- First column I figure 4 shows the percentage distribution of CI IA "positive" primary carcinoma cell cultures, - as measured on micro-slides, which had been incubated with the antibody in a Leighton tube in a humidified 37°C C0 2 incubator.
- the percentage CI IA positive carcinoma cells in the cell cultures were plotted into the diagram. It appeared that around 25% to >75% of the carcinoma cells were found positive.
- Second column of the figure shows the distribution of carcinoma cell cultures sub-cultured in Falcon flasks and re-explanted on micro-slides in Leighton tubes. These measurements showed approximately the same distribution in percentage of CI IA positive cells.
- the third column show carcinoma cell cultures pre-incubated with unconjugated polyclonal rabbit anti CI IA, then washed times 3 in PBS buffer and re-incubated with FITC-conjugated rabbit anti CI IA. No cells showed any specific excitation.
- the fourth column incubated with FITC conjugated rabbit anti human CI IA and measured as described above showing no specific excitation when measured with the above described Cytophotometer indicating no specific binding of anti CI IA on these non-malignant cells, which included among others human mesenchymal cells.
- the cells used for this measurement were explanted on micro- slides, subjected to 30 minutes incubation at in a C0 2 incubator at 37° C with FITC conjugated poly anti CI IA (BehringWerke, now Behring Institute), or initially incubated with un-conjugated rabbit polyclonal anti human CI IA and thereafter with FITC conjugated anti CI IA and measured as described above.
- FITC conjugated poly anti CI IA BoehringWerke, now Behring Institute
- the percentage of carcinoma cells binding FITC conjugated rabbit anti CI IA ranged from around 25% to around or >75 % of the cells of the total of 100 cells measured from each culture.
- the binding of the FITC conjugated anti CI IA demonstrated according to the inventor a specific binding of anti CI IA to the plasma cell membrane of these malignant cell types, based upon the pre- saturation assay, where the carcinoma cells were pre-incubated with unconjugated rabbit anti human CI IA, followed by 3 times of washing in PBS at pH 7.2-7.4, and re-incubated with FITC conjugated rabbit anti human CI IA from the same manufacturer (BehringWerke, Marburg, Germany).
- Non- malignant cell types consisting of mesenchymal human cell lines did not show any specific binding of FITC conjugated rabbit anti CI IA under the exact same condition as the carcinoma cell lines, (see Figure 4).
- This infant was a 28 months old male with a malignant metastatic
- the first testing of blood CI IA was done at this time. A high level of CI IA, around 85 mg/100 ml was found (normal range 15 to 35 mg/100 ml).
- CI IA Blood testing for CI IA showed an initial immediate increase to around 90 mg/100 ml, which was theorized to be caused by lysis of cancer cells. Five (5) days after the infusion CI IA decreased to 50+ mg/100 ml and C4 decreased from between 50 mg/100 ml (theorized normal range 20-40 mg/100 ml) to 20+ mg/100 ml.
- Vincristin treatment had to be stopped at third dose because the patient re-developed neurotoxic symptoms, ataxia and paresis, but since this treatment, no serious infections were noticed.
- IgG B lymphocytes The high previously high level of IgG B lymphocytes declines significantly prior to anti CI IA infusion. An increase in IgG B lymphocytes increased
- the patient had previously been treated with irradiation against bilateral supra, infra and axillary lymph nodes, and palliative irradiation against lumbar column.
- the patient was initially infused slowly with xenogeneic (porcine) semipurified IgG anti human CI IA purified from pigs immunized with semipurified CI IA.
- the patient was infused slowly with an initial total dose of 680 ml (a 10% porcine IgG concentration) after testing negative to cutaneous test for porcine immunoglobulin. No significant temperature rise occurred.
- the patient received two cycles (one week interval) cytostatic treatment with Vincristine (total dose 1.15 mg), 5- fluorouracil (total dose 600 mg), metothrexate (total dose 45 mg),
- cyclophosphamide total dose 150 mg
- prednisone total dose 550 mg
- semipurified anti CI IA a second and a third xenogeneic dosage was given each time with approximately 10% xenogeneic sheep antibody, because the patient showed positive skin test to porcine proteins, and negative for sheep protein.
- the semipurified IgG anti CI IA obtained from sheep of type Oxford after the animal had been immunized and boosted with semipurified CI IA.
- This xenogeneic antibody was administered over two days with an interval of 7 days (total dose of the two cycles 580 ml with a immunoglobulin concentration of 10%). Maximum temperature rise 40.4. degree. C, which reverted after some hours to normal temperature level.
- metastases flattened, and changed to horny scars.
- the osteolytic metastases were after a period of 3.5 months replaced by osteosclerotic structures.
- One bone marrow sample showed normoplastic marrow, another showed small islands of tumor cells surrounded by lymphocytes and connective tissue, and normoplastic marrow.
- the patient started out with a serum level of CI IA of approximately 82 mg/100 ml (normal range 15 to 35 mg/100 ml) and a serum native C4 of 58 mg/100 ml (theoretical normal range 20-40 mg/100 ml).
- a serum level of CI IA of approximately 82 mg/100 ml (normal range 15 to 35 mg/100 ml) and a serum native C4 of 58 mg/100 ml (theoretical normal range 20-40 mg/100 ml).
- serum CI IA of approximately 82 mg/100 ml (normal range 15 to 35 mg/100 ml)
- a serum native C4 of 58 mg/100 ml (theoretical normal range 20-40 mg/100 ml).
- the level of serum CI IA increased to around ⁇ 95 mg/100 ml and native C4 increased to ⁇ 65 mg/lOOml.
- serum CI IA decreased to around 82 mg/lOOml
- Vincristine serum native C4 decreased to around 40 mg/100 ml. After the first two cycles of chemotherapy incl. Vincristine serum CI IA decreased to 55 mg/100 ml and was at the same level, when starting the second series of xenogeneic semipurified administered over two days anti CI inactivator, the CI IA immediately increased with two distinct repetitive increases followed by a gradually decline of CI IA,
- the thrombocytes dropped during the combined cytostatic treatment and INA treatment concomitantly to the development of purpura, treated with transfusions. Furthermore, a developed sinus tachycardia was observed and treated with excellent effect.
- carcinoma cell cultures tested for the presence of CI inactivator as described previously represents a certain number of the cells using rabbit anti human CI inactivator-fluorescein isothiocyanate (FITC) labelled and tested using immunofluorescence microscope as described.
- the immunoflurorescence was measured arbitrarily by a Leitz MPV 1
- Cytophotometer built on a Leitz Orthoplan phase contrast microscope with measuring window which could measure one cell at a time, the result of which was written down. A number of 100 cells were brought into the diaphragm, one at a time, and the excitation value was written down. Controls was done by measuring cells incubated in Leighton tubes, starting with initial incubation of the cells in unlabeled rabbit anti human CI IA for 30 minutes at 37°C C0 2 incubator, washed times three in PBS buffer and then incubated with anti CI inactivator-FITC, the cells on the microslides were then washed x 3 with PBS, and measured using the Leitz MPV1 Cytophotometer microscope.
- the cancer cells on the new microslide was cultured for approximately 3 days, and incubated with anti CI IA-FITC, a various percentage of the cancer cells were again binding anti CI IA-FITC indicating that the cells had reproduced the coating of CI IA on their plasma membrane.
- This measurement was performed in several carcinoma cell cultures from various types of carcinomas from various organs, from pleura fluid or ascites fluid drawn from patients suffering from metastatic carcinomas of breast, or from abdominal organs.
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Abstract
The present invention pertains to a method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic to which the membrane, an immortalised cell from the amniotic fluid, HEK cell lines and a unique HEKts cell line, which works optimally with the vector system, developed by HumanCell Co. (HumanCell Co. Naperville, Illinois, U S A), CAP cell lines and CAP-T cell lines, also disclosed are a human host cell encoding the CI esterase inhibitor (CI IA), a recombinant CI esterase inhibitor (CI IA), a composition comprising the recombinant CI esterase inhibitor (CI IA), use of the recombinant CI esterase inhibitor (CI IA), an antibody raised against the recombinant CI esterase inhibitor (CI IA), a method for detecting and/or screening and/or monitoring cancer in an individual and a method of diagnosing rejection of transplants.
Description
Recombinant CI esterase inhibitor and use thereof
Technical field of the invention
The present invention relates to a method a method for the production of recombinant CI esterase inhibitor (CI IA), a human host cell encoding the CI esterase inhibitor (CI IA), a recombinant CI esterase inhibitor (CI IA), a composition comprising the recombinant CI esterase inhibitor (CI IA), use of the recombinant CI esterase inhibitor (CI IA), an antibody raised against the recombinant CI esterase inhibitor (CI IA), a method for detecting and/or screening and/or monitoring cancer in an individual and a method of diagnosing rejection of transplants as well as a method using a human or humanized antibody raised against the recombinant CI esterase inhibitor for repeated treatment of certain cancer types with said antibody.
Background of the invention
In US 4,132,769 was filed for the use of a suspected blocking protein that he named CI IAC, which was semipurified using various known purification methods wherein one of the other proteins appeared to be CI esterase inhibitor or CI inactivator. Several years later, it appeared after additional purification methods that the suspected blocking protein, CI IAC, actually showed to be another acute phase protein, C-Reactive protein (CRP). The antibody containing antigen determinants against the semipurified proteins was tested on cancer cells and malignant brain cells and the main part of the semipurified protein CI inhibitor also called CI esterase inhibitor or CI inactivator, towards which the immunoglobulin fraction from xenogeneic animals (porcine antibody) was produced.
Previous description of patients treated with porcine sermipurified anti CI inactivator
This treatment was described in e.g. US Patent 4,132,769. The xenogeneic (porcine) antibody could only be used once or at the highest twice, after which the patients got immunized against porcine proteins, preventing any further anti CI IA treatment at that time, and thus the experiment now has and had only academic meaning, until one could find a manner of providing a human or humanized antibody against CI IA, which was not possible at that time at all.
The pig proteins at that time was thought to resemble human
immunoglobulins, which was used to treat a few patients with advanced metastatic cancer utilizing the xenogeneic antibody against the semipurified proteins. It appeared that the xenogeneic antibody decreased the tumor load in these few patients and at the same time when measuring CI inhibitor or CI inactivator using rabbit anti CI inhibitor antibody against CI inactivator (from BehringWerke) and C4 (from BehringWerke, now CSL Behring) the treatment also revealed a decline in CI inhibitor and in C4.
However, this approach to immune treatment against cancer was so new and because it could only be used as a single application or at the highest 2 applications tested in a few patients. After this first or second injection, cutaneous testing showed that the patient produced antibodies against pig proteins, and due to this appearance of possible immune reaction prevented further treatment, said treatment approach accordingly ended up only as being perceived was at that time was only perceived as having academic interest.
Additionally, the main interest among oncologists at that time was the significant focus in developing various chemotherapy drugs and combining this form of therapy in complex protocols in order to decrease the toxic effect of "one" chemo-drug, so that the concept was to spread and may be lessen the toxicity of the various approaching chemotherapeutic derivates, and because the point of attack against the cancer cells could be as multifaceted as possible, the protocol developed at that time are still used as the current treatment approach.
Of course combined with the approach where the physicians had the longest experience, namely surgical intervention. Since that time it is still the chemotherapeutic protocol approach with multiple approaches that more or less is the golden standard treatment. A few other approaches has been included, whereof Osther was involved for some years in the interferon alpha treatment added to the chemotherapy protocols. In the recent years monoclonal antibodies added to the chemotherapy protocols have not improved the survival rate significantly.
Hence, an improved development in the area of more sophisticated
immunologic treatment, and the tools generated to produce various proteins as recombinant synthetic proteins, the possibilities to create humanized or even human proteins, has paved the way towards producing immunoglobulins that can be tolerated well in patients and which now can be injected and/or infused in patients repetitively where the patients do not produce or in a lesser degree produce antibodies against. The advance in the understanding of molecular genetics has given us tools with which we can dig deeper into the behaviour of various immune defence systems and again focus on significantly improve the possibility of creating for instance antibodies I now more than ever believe will provide us with tools that can efficiently attack certain types of cancers using for instance the complement system as an extremely potent system towards eliminating cells, such as for instance certain cancer cells, as long as we build further on our know how and our possibilities now a days towards profoundly changing for instance the behaviour of cancer cells from making themselves invisible to give us remedies where the cancer cell suddenly has to be visible and thereby vulnerable to combined immunological and complement system attack, which at the same time provide us with remedies to easily follow up the effect on cancer among others with simple measurement of the molecular changes that renders the otherwise invisible cancer cells visible and vulnerable, towards a
disclosure of the cells that enable us to fight them by removing one of their hiding mechanisms.
Summary of the invention
Thus, an object of the present invention relates to the surprising findingCl esterase inhibitor (CI IA) and native C4 can be used in a method for detecting and/or screening and/or monitoring cancer in an individual.
Thus, one aspect of the invention relates to a method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines.
Another aspect of the present invention relates to a method for producing recombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI esterase inhibitor developed at the end of the Fab or (Fab)2 of the immunoglobulin. The anti CI esterase inhibitor "paratope" (analogous to a lock), which is located at each tip of the "Y" of an antibody. This paratope is specific for one
particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision.
Yet another aspect of the present invention is to provide human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the human cells such as HEK cells, PerC6 cells, amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a CI esterase inhibitor.
Still another aspect of the present invention is to provide a recombinant CI esterase inhibitor obtainable by the method above.
A further aspect the present invention pertains to a composition comprising a recombinant CI esterase inhibitor, characterised in that
i) said composition comprises substantially no synthetic protease inhibitors, and/or
ii) said composition is serum free. Still another aspect the present invention pertains to a recombinant CI esterase for use as a medicament.
In yet another aspect the present invention pertains to a method for preventing, treating and/or alleviating Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI esterase inhibitor.
In another aspect the present invention pertains to a method for treating burn wounds, Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor.
In a further aspect the invention pertains to an antibody raised against a CI esterase inhibitor.
In another aspect the invention pertains to a method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
a) a first parameter represented by the concentration of CI esterase
inactivator (CI IA) in at least one excreta from the individual and b) a second parameter represented by the concentration of complement component native C4 in at least one excreta from the individual
wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
In another aspect the invention pertains to a kit for performing the above method, wherein said kit comprises CI IA and native C4.
In yet an embodiment the invention pertains to a method of diagnosing rejection of transplants, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
b) incubating excreta from a human with anti-C4 and determining the
native C4 level in said sample
c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4 level with a second reference- level e) determining whether said human is likely of having cancer if the level of Cl-IA is at or above the first reference-level and if the level of native C4 is below the second reference-level.
In an aspect the invention pertains to a method of diagnosing cancer, said method comprising the steps of
(a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
(b) incubating a blood sample or exsudate or transudate in connection with a suspected rejection of (transplanted) tissue from a human with anti-C4d and determining the C4d level in said sample
(c) comparing the determined CI IA level with a first reference-level,
(d) comparing the determined C4d level with a second reference-level (e) determining whether said human is likely of experience a tissue or bone marrow rejection if the level of Cl-IA is normal or lower than 15 mg/100 ml and if the level of C4d is higher than 50 mg/100 ml.
Detailed Description of the invention
Previous description of patients treated with porcine sermipurified anti CI inactivator
The first attempt to use antibody that at that time was described in US Patent 4,132,769 as a cancer derived CI inhibitor, or cancer derived CI inactivator, where based on the methods ivailable in the mid 70ies made it difficult to clearly discriminiate between proteins using the more primitive immunological remedies at that time. At that time the inventor of US Patent 4,132,769 was sure that there were two different CI inactivators, namely one type that was produced by cancer cells, called CI IAC, and another one found in patients as such, which was CI IA. One of the proteins appeared to the invenor at that time to be constituting the CI IAC appeared in the late 80ies and 90ies to be C reactive protein that had called the confusion between cancer derived CI inactivator and the CI inactivator (also called CI inhibitor, or CI esterase inhibitor). When using the semipurified anti CI IA, at that time believed to constitute CI IAC that could cross react with CI IA. The method using what the invenor now know was anti CI IA was based on using porcine
immunoglobulins. These xenogeneic (porcine) antibody could only be used once or at the highest twice, after which the patients got immunized against porcine proteins, preventing any further anti CI IA treatment at that time, and thus the experiment had only academic meaning.
It is an extremely important part of this invention to enable a neutralization of the CI IA coat on the cancer cells in the types of cancer described in this patent application. However, as stated it did not make sense to further the possibilities with the use of porcine immunoglobulins. The inventor and his research team tried as late as from 1995 to 1998, tried to genetically modify the heavy and light chains of IgG attempting to preserve the hinge region, and tried to make it possible to produce antigenic determinants against proteins such as CI inactivator, but we did not succeed in humanizing the pig immunoglobulins.
Furthermore the inventor and his team worked together with Tufts University Veterinarian School to try to clone pigs, where they had had some luck in changing the skin on pigs towards human skin, but the cloning did not materialize in a humanization of the pig to a degree, where it would produce human or humanized proteins, so that meant that the invenor had no way in that manner to perform more cloning of pigs in attempts to humanize these animals' protein production, mainly also because it would take many years and it would be impossible to raise sufficient capital to finance such study, that did not show any luck within these years in the mid 90ies.
An attempt to produce another proteins such as antibodies in pigs against tumor necrosis factor alpha also was tested by the inventor and his team in the mid 90ies, where it was actually seen that when patients with severe rheumatoid arthritis was injected with 10 gram of porcine antihuman Tumor Necrosis Factor alpha, their symptoms, such as joint morning stiffness, pains swelling of their joints, disappeared within 10-12 days, but, again
unfortunately , the treatment could only be given once, and no other possibilities was found at that time to get such efficient treatment with monoclonal antibodies as the very efficient porcine anti human TNF alpha.
Therefore, the most important goal in the opinion of the inventor is, to identify, develop, and produce an efficient antidote to neutralize CI IA coat and at the same time balance the treatment in such a manner that the necessary amount of CI IA in circulating blood, the lymphatic system and other body fluids including the necessary amount of CI IA in the CNS system to avoid the treatment to close down the CI IA production to a level, where the "classical pathway" for complement can still be active in order to avoid that the patient develop an angioedema also called HANE-like condition. There may already be biopharmaceutical or other biological product - approaches that can inhibit CI IA or its inhibitory effect. Even though
Aprotinin is not in the focus as an inhibitor of CI inactivator, and even though Aprotinin is a xenogeic protein, there is a possibility that those researchers
working with Aprotinin might produce a recombinant anti Aprotinin that in theory could inhibit CI inactivator activity; for instance Wachtfogel et al described that for instance Kallikrein-Cl-inhibitor complexes were completely inhibited and Cl-Cl-inhibitor complexes were partially inhibited
at aprotinin concentrations of 0.03 mg/ml or greater (Wachtfogel YT, The J. Thoracic and Cardiovascul. Surg. (1993)106(1) : 1-10). in the future be developed drugs that can inhibit CI IA in humans including inhibiting CI IA inhibitory effect on cancer cells. There are several ways the CI IA coat on cancer cells could be attacked. One way is to directly attack the CI IA with a very potent anti CI IA antibody that at the same time could neutralize the inhibiting activity of CI IA focused on primarily targeting the cancer cells.
Such approach could in theory be approached by obtaining blood from a patient with angioedema associated with a B-celllymphoproliferative disorder that became evident 9 months after C1INH deficiency was diagnosed, and androgen therapy stopped the attacks of angioedema. This category of patients with lymphoproliferative disease and angioedema has been described previously (Frigas E. Mayo Clin Proc. 1989 Oct;64(10) : 1269-75). Cicardi et al found at the department of Internal Medicine, University of Milan, Italy, among 23 patients through some years with angioedema. Autoantibodies against CI inactivator was present in 17 of these patients, and in some of these patients were 3 patients with (Non Hodgkins) lymphomas, and one patient with chronic lymphatic leukemia (Cicardi M, Zingale LC, Pappalardo E, Folcioni A, Agostoni A. Autoantibodies and
lymphoproliferative diseases in acquired Cl-inhibitor deficiencies. Medicine (Baltimore). (2003) Jul;82(4) : 274-81).
B lymphocytes could be harvested from for instance 50 cc of fresh blood, B lymphocytes (whereof some of these will produce anti CI inactivator) could be isolated by using Hypaque Ficoll centrifugation. These B lymphocytes could be cultured and the anti CI inactivator secreting B lymphocytes could be caught by immobilized CI inactivator using a cell sorting equipment. These B cells could then be proliferated and the immunoglobulin producing antigenic
determinants against CI inactivator could be isolated, and the antigenic determinants of these immunogloubulins could be subjected to molecular biological isolation of the antigenic determinants, and the code for the anti CI inactivator could be disclosed. This code could then be the basis for the production of recombinant human IgG (or IgM) with binding specificity to Clinactivator. For instance in this manner a human recombinant anti human CI inactivator, which could isolated, purified and used for the infusions in humans without risking any significant allergic or immune reaction in patients. In this manner cancer patients with carcinoma cells or other malignant cells with CI inactivator coat, could then be treated repeatedly, and of course monitored by measuring the blood CI inactivator and C4, to balance the treatment with anti CI inactivator by following the balance, so that the patient can be safely treated without falling below the until further postulated low levels of blood CI inactivator and blood C4. One could then get sufficient experience in treating such cancer patients until the patient either went into remission, or at least one might obtain a partly remission and thereby in theory prolong the life of the cancer patients using this approach.
Apart from the described approach above, the theoretically most ideal antibody would be a human or humanized anti human CI IA antibody that at the same time can neutralize the CI IA activity and secondly alert the initiated antigen - antibody reaction against CI IA at the same time could activate the "classical pathway" of complement, meaning an immunoglobulin of type as for instance IgG (minus IgG4) or IgM that at the same time would cause disassociation of the Clqrs complex to a degree, where C4 is activated and start the binding to the surface of the cell which again would activate C3, C5 all the way to C9 where these cumulative bindings would end up producing "pores" on the cancer cell and thereby lysing the cell. At the same time will all the subunits of the various activated complement factors elicit subunits of many of these molecules that will cause opsonin effect, attracting various cells such as macrophages neutrophils, and cells originated from the immune system, to in a concerted effort would start
removing the attacked and probably damaged or lysed cancer cells. At the same time, the antibody most ideal for this purpose would also be capable of being repeatedly infused to the patient during months may be years to obtain the most efficient kill of the cancer cells.
Another approach of the specific human, preferably recombinant or humanized monoclonal antibody with high binding capacity being capable of carrying either a cancer-killing component, may be in a form of a potent
chemotherapeutic agent which otherwise could be given systemically without producing potent and serious side effects. Another approach could be to bind a radioactive emitter to the cell for direct cell killing amount of radioactive doses, such as for instance a beta emitter with relatively low spreading to surrounding tissue.
Another approach would be to bind a tracer to the specific anti CI IA antibody, which could be Technetium-99m is a metastable nuclear isomer of
technetium-99 (itself an isotope of technetium), symbolized as 99mTc, that is used in tens of millions of medical diagnostic procedures annually, making it the most commonly used medical radioisotope. Technetium-99m is used as a radioactive tracer and can be detected in the body by medical equipment (gamma cameras).
The importance and the schematic mechanism of the Anti human
(mammalian) CI inactivator (CI IA) in human antibody, either as humanized monoclonal antihuman CI IA or as human recombinant anti human antibody , as IgG or IgGl or IgG3, to be used parenterally for the treatment of cancer of type Carcinoma, malignant brain tumors such as Astrocytomas/glioblastomas, or certain types of malignant Sarcomas benefitting patients with primary cancers or metastases
The Importance of the anti CI IA recombinant human IgG or IgM (minus IgG4) treatment of cancer patients with coated CI IA cells.
In order to succeed in producing recombinant antihuman antibody from human IgG, this can be done in various cell types found optimal for
transfection of human IgG or subclasses of human IgG such as IgGl, or other subtypes of IgG.
The primary goal of an anti human CI IA antibody is that one can repeat the treatment, unlike what one can do with xenogeneic antibodies, which historically indicated to have tumor regression effect, but at the same time, only academic interest, because one could only administer the xenogeneic antibody once or twice, otherwise the patient showed immunogenic reaction against the xenogeneic antibody, and prevented further treatment even though one could demonstrate effect in regards to tumor regression in patients treated with this anti CI IA.
A continuous treatment over weeks, months and years can be done, when the human does not produce antibodies to the exogenously applied humanized monoclonal anti CI IA or even better applying a recombinant human anti CI IA (e.g., preferably IgGl) and at the same time follow the decrease of the increased CI IA found in cancer patients circulating in the blood proving to be an excellent way of indicating effect at the same time as the cancer
diminished in size. This could only be followed for weeks to months, because more anti CI IA treatment was needed, but could not be done with the technology at that time.
New ways had to be invented, and this actual invention described herein shows the way of producing an anti CI IA antibody that can be repeatedly administered to cancer patient as often as needed, the more human like, humanized monoclonal anti CI IA or even more optimal by using recombinant human IgG and preferably of type Human IgGl (or other IgG types, except for IgG4 which is not related to or working with the complement system. All the other types of IgG and actually also IgM would be optimal because these immunoglobulins induces the "classical pathway". It is the classical pathway exemplified in figures 4a, 4b, and 4c that decides the involvement of the complement system during antigen-antibody reactions on cells.
Based upon the relatively few previous studies described in Osther patent (US patent 4,132,769), it is difficult to identify an animal model to use for this purpose, I administered between 180 to 300 ml (10% IgG concentration) of xenogeneic semipurified anti CI IA as shown on figures 4 and 5 on two patients treated with xenogeneic sermipurified (porcine) anti human CI IA, where the CI IA and in some cases, C4 was monitored in the blood (see figures 4 and 5). The graphs show the fluctuation of CI IA and C4 over several weeks after one to two IV infusions of xenogeneic (porcine)
semipurified antihuman CI IA.
Figures 4 and 5 present serum CI IA and serum native C4 (meaning these proteins circulating in the blood) during the treatment of a 28 month old infant with Xenogeneic (porcine) polyclonal anti human semipurified IgG fraction of antibody from pigs immunized with semipurified CI IA.
Native C4 any other native protein is to be understood as the native state of a protein or nucleic acid which its properly folded and/or assembled form, which is operative and functional. Most human cancer types (~85% of all cancers) are carcinomas and these types of cancers have shown the presence of an inhibitor of the complement system, that the inventor have found, is the CI inactivator (also called CI inhibitor or CI esterase inhibitor). This type of cancer that appears to exhibit the CI inactivator (C1IA) on their plasma membrane are derived from epithelial cells that arises in the embryon from cell in the endodermal or ectodermal germ layer. It is postulated that these types of cancers occur because of altered DNA in these cells. Furthermore, the inventor have also indicated that certain malignant brain tumors such as astrocytomas,
glioblastomas, medulloblastomas and other types of primary brain tumors, by testing some of these primary brain tumors with rabbit anti human CI inactivator-FITC conjugated, also carry this inhibitor.
First of all it is important to describe the difference between the carcinomas and the malignant brain tumors supposedly ability to produce CI inactivator and present this protein on their plasma membrane. The primary malignant brain tumors, when cultured in cell culture flasks, have shown that they after one or more sub-culturing processes lose the ability apparently to produce CI inactivator and present this on their plasma membrane. This is significantly different from the carcinomas, which in subcultures repeatedly reproduce the CI inactivator (or CI IA) on their plasma membrane, even when carcinomas have metastasized to the brain (thus being sometimes described as secondary brain cancer).
The definite difference between the carcinomas and the primary malignant brain tumors is postulated by the inventor to be caused by the fact - that it is known that primary brain tumors (even though they are sufficiently
vascularized and thereby in connection with the rest of the body's blood circulation, they do not produce metastases outside the central nervous system. Thus, unknown factors in the Central Nervous system (CNS) are participating in keeping the malignant brain tumor cells alive and allowed to proliferate inside the CNS, - factors that apparently do not exist in the body outside the CNS system. It cannot be an immune reaction that is due to this difference, because the brain cancer cells lose this ability in cell cultures, where they are not in contact with any immune system in the medium.
The brain tumors are accordingly protected against the complement cascade as long as they are retaining their CI inactivator on their plasma membrane, but are apparently loosing this ability to produce more CI inactivator for protection when sub-cultured, and therefore the primary brain cancer cells will either lack the ability to grow outside the CNS, because of unknown factors keeping them alive in the CNS, or if they are released into the blood
circulation and exposed to the condition of the rest of the human body, one could postulate that the condition outside the CNS may differentiate the cells as has been observed in the microscope when studying subcultured CNS brain cancer cells, because their appearance change towards resembling fibroblast-
like cells and as discussed above, lacking the CI Inactivator coat on their plasma membrane.
This difference between brain cancer cells and carcinomas enable the carcinomas to metastasize anywhere in the body including into the brain, and therefore are not dependent upon some specific "non-CNS" environment.
The unique coating of certain human cancers (other than malignant brain tumors), especially carcinoma. Carcinoma is a type of cancer that develops from epithelial cells. Specifically, a carcinoma is a cancer that begins in a tissue that lines the inner or outer surfaces of the body, and that generally arises from cells originating in the endodermal or ectodermal germ layer during embryogenesis. Carcinomas are the most common type of cancer. They make up about 85 out of every 100 cancers in the UK (85%).
Thus, anti CI inactivator would first of all according to the inventor be one major important reason for cancer to evade the body's humoral immune defense system (which again normally interact with the cellular immune system at many levels) by simply blocking the complement activation already at the first complement component, the Clqrs complex where the normal dissociation of this complex would result in activation of the full C4
complement component for it to dissociate and bind to the target cell surface of the same cells that inhibits the Clqrs complex to be activated.
There are three ways according to the inventor that this surprising appearance of CI inactivator on cells can be utilized.
1. As Cancer in vitro diagnostic (by measuring CI inactivator in blood or body fluids (and native C4 complement component, see explanation later)
2. As Cancer in vivo diagnostic and tracer (e.g., a radioactive isotope by labelling a humanized antibody against CI inactivator.
3. As Cancer treatment agent, such as anti human or anti humanized CI inactivator
4. As Cancer human or humanized anti CI inactivator coupled to a anti cancer treatment drug, which e.g., could be delivered directly to the target cells
This invention provides a unique and novel way to side-track the protecting CI inactivator coat, by introducing a recombinant human IgG antibody having antigen binding sites against CI inactivator.
There are three ways to approach anti CI inactivator antibody.
One of the unique strategy I chose was to identify a polyclonal antibody of IgG type in those patients that had inherit anti CI inactivator antibody, and the antibody would be an IgG, or even an IgM. Among those patients I selected to identify patients or individuals who have CI inactivator deficiency or who lacks CI inactivator (HANE, or Quincke' edema, also called angioneurotic edema) due to an endogenous production of antibody against CI inactivator
Another way was to identify patients with certain lymphoproliferative diseases or lymphomas who produce are known in some ways to develop autoimmune antibodies (e.g., antibody to CI inactivator.
So, fresh blood from patients with these disorders (e.g., 50 - 100 ml would suffice), and the B lymphocytes be isolated by using IsoPaq- Ficoll
centrifugation and removing the thin layer of lymphocytes. Catch the B lymphocytes that produce anti CI inactivator by cell sorting, and isolate and select the genes for Anti CI inactivator immunoglobulin from these
lymphocytes, and for instance by using molecular methods to detect the appearance of the paratope directed against the CI inactivator. Such diseases as acquired angio-oedema; - ymphoproliferative disease;
- autoantibodies
The inventor looked for autoantibodies to CI inhibitor (Cl-INH) and evaluated the relationship of their presence to the associated lymphoproliferative diseases and to the cleaved form of Cl-INH in 13 patients with acquired CI-
INH deficiency (acquired angio-oedema (AAE)). At the time of manifestation of angio-oedema symptoms or within a few years the following diseases were diagnosed : liver angioma {n - 1), M-components {n - 7, one of whom also had echinococcal liver cysts), breast cancer (n- 1), chronic lymphocytic leukaemia (CLL;n= 1) ; three patients had no associated disease.
Anti-Cl-INH autoantibodies, measured both as immunoglobulin binding to Cl- INH immobilized onto microtitre plates (ELISA) and as plasma inhibitory activity of Cl-INH function, were found in 12 patients. Binding of Cl-INH to paraproteins, transferred to Immobilon after agarose gel electrophoresis, was detectable in five of seven M-components associated with AAE.
Immunoblotting analysis of SDS-PAGE-separated plasma demonstrated that Cl-INH circulated in the cleaved 96-kD form in the 12 patients with
autoantibodies, but not in the one without. In conclusion, the large majority of our patients have autoantibodies to Cl-INH . Circulating autoantibodies are necessary for the generation of cleaved Cl-INH . The paraproteins associated with AAE are frequently autoantibodies to Cl-INH and thus account for its consumption .
The white blood cells normally work together with the humoral immune system activated complement components to attack any foreign invaders, or foreign cells, so the immune system as such will in its major defence system utilize both the cellular, and the humoral immune system in its elimination of foreign or allogeneic cells. Even though carcinoma cells in some way should be postulated to be recognized as "foreign" cells, they are protected to a degree that does not allow the body to get rid of these cells, which I would postulate could be called "protective mutagene" cells, because the difference is most probably a mutation in the DNA which has changed the "protected - normally called malignant cell".
If the body could recognize these cells as "unprotected mutagene cells" instead of protected cancer cells, the body would most probably eliminate these cells that in a non-protective stage via the immunosurveillance system because now the so-called malignant cell (after protection has been stripped from the cell) would be detected as being non-self, and the immune system
including the complement system would then eliminate cells as if it was an allogeneic cell, and one could postulate, that if one changes the protective malignant cells and render them unprotected, the body would attack these cells as were they "pseudo-autoimmune cells". Even though so many protein differences have been shown to be present in certain carcinoma cells and not in the normal non-malignant epithelial cell lines in the body, the body will not recognize them as foreign in a manner that might eliminate them. Of course it is impossible in advance to postulate that the body cannot get rid of malignant cells, when they appear at first, which has been postulated by many authors that man probably is attacked many times in life and in theory may fight off small attempts to produce to called malignant cells with the immune system.
However, then it is a semantic matter whether the cells might be defined as malignant, as long as the body can recognize these cells. May be they should be viewed as a form in a way undistinguishable from "allogeneic" cells that might be protected by some mechanism, which would not recognize them as allogeneic - due to a theoretical protective system, which in theory might appear in the human body. That is the reason for the inventor to call these protected malignant cells for "protected mutagenic cells", and therefore, one could postulate that what one previously viewed as malignant cells, that the body could get rid of itself, actually from an immunological stand point do not turn malignant before they are capable to protect themselves from the immune surveillance system or turn into a cell capable of escaping the immune system or blocking the immune system from attacking the cells, and at that time it would or should be called malignant.
The criteria "malignant" could thus appear when the immune system cannot recognize or according to this invention is inhibited from using its immune system fully because of inhibiting factors appearing in the cell that now has turned malignant. So, the malignant cell could also be called a protected cell that can freely spread to any tissue in the body and often.
From this view point the malignant cells could also be viewed from the perspective of protection, where the protection is a protein such as CI inactivator, which is not recognized as foreign for the immune surveillance in the body, and therefore, the immune system would not attack the cells using the complement system as eliminator of these cells. Because it appears from figures 1 and 2 that a substantial percentage of malignant cells (e.g., carcinoma cells) show coating of CI IA as described in Example 1, the blood shows increased amount of CI inactivator, when compared to patients or individuals with non malignant disease, and therefore it is part of this invention to use an specific anti CI inactivator antibody to identify cancer (e.g., carcinoma) activity in humans.
There are a few conditions where circulating CI inactivator would appear increased other than in cancer. Therefore it is necessary to at the same time measure the presence of the "full" C4 complement component, which would be increased in patients, where C4 is blocked from reacting with Clqrs, due to the presence of increased amount of CI IA, which in case one also find an increased C4 component, a complement blocking is going on in the patient at the level of Clqrs, and it would be possible that this blockage is due to the production of CI inactivator from for instance the cancer cells, which at the same time would block the C4 component, from splitting to C4 sub units. So when comparing Example 1 and figure 1 with Example 2, figure 4, the increased circulating CI inactivator and the blocked full C4 component found in figure 1 could explain that cancer patients apparently have increased circulating CI inactivator and full C4 component in the blood. This has to be held together with the findings of CI inactivator coating the cancer cells as shown in Example 2, figure 4.
Thus, it is within the scope of this invention to combine a cancer detecting and cancer monitoring kit, a dual detection kit, consisting of highly purified antibodies with high specificity to the CI esterase inhibitor also described as CI inactivator (CI IA) and the and complement component native C4, and certainly not C4a or C4b.
Native, human C4 complement component is a glycoprotein composed of three non-identical subunits of M.W. 93,000 (a), 75,000 (β), and 32,000 (y) linked by disulfide bonds. Present in normal human serum at 400 pg/ml or 40 mg/100 ml. On activation of complement via the classical pathway, the Cls subcomponent of the CI complex is converted to an active serine protease that cleaves the C4 a-chain at peptide bond 77, resulting in the production of C4a (M.W. 8740) and C4b fragments (M.W. 193,000). The released C4a peptide is one of the three complement-derived anaphylatoxins. The nascent C4b fragment can form a covalent ester bond with target surfaces. This covalent attachment of C4b to target acceptors is required for continuation of activation via classical pathway.
The breakdown of native C4 (present in normal human serum at around 40 mg/lOOml) to C4b predicts activation of complement via the classical pathway, the Cls subcomponent of the CI complex is converted to an active serine protease that cleaves the native C4 resulting in production of C4a and C4b fragments. C4a peptide is one of the three complement-derived anaphylatoxins. The nascent C4b fragment can form a covalent ester bond with target surfaces. This covalent attachment of C4b to target acceptors is required for continuation of activation via classical pathway.
In order to establish the blocking of the complement system in the classical pathway, due to the large presence of CI IA on cancer cells, reflected in elevated blood concentration of CI IA. This would hinder the the native C4 from breaking down to C4a or C4b subunits. It is therefore according to this invention important when this cancer diagnostic and prognostic "dual test kit" besides measuring the level of CI IA (normal range in blood 15 to 35 mg per 100 ml) certainly measure the level of native C4 (normal range in blood is approximately 40 mg per 100ml).
As indicated, carcinomas are the most dominant malignant organ related tumors where the cancer appears to escape the human immune defence system including the complement system, where the classical pathway for a
complement cascade, normally related to an antigen-antibody reaction on the surface of a pathological cell or microorganism.
Future CI inactivator and naive C4 testing systems
In the future the dual kits for routine testing will be using two different methods, one being a measurement using immune testing apparatus, such as for instance ELISA readers using conugated recombinant anti human CI IA, conjugated recombinant human C4 or conjugated recombinant C4d for example using Thermo Scientific ELISA equipment. In regards to the evaluation of a cancer diagnostic according to this invention is that, besides measuring the CI IA, the other complement component to be measured in the cancer diagnostic kit according to this invention is the "intact" molecule, the native C4. If both CI IA and C4 is elevated, it is according to this invention indicating the presence of active malignant cancer tissue. The subunits C4a and C4b does not play a role in this diagnostic kit, but would be more aimed at measuring C4b or other subunits in patients with autoimmune disorders (Mortensen S, Kidmose RT, Petersen SV, Szilagyi A, Prohaszka Z, Andersen GR, J Immunol. 2015 Jun l; 194( l l) : 5488-96). Another subunit of C4 is called C4d is reported to be increased in tissue from liver transplant patients showing signs of acute liver rejection or ongoing full acute or chronic liver rejection, so in such case to evaluate the rejection mode in the organism, for instance liver rejection, a C4d measurement would be useful (Gierej B, Kobryh K, Gierej P, Gornicka B, Ann Transplant. 2014 Aug 1; 19 : 373-81). It can of course not be excluded - and may even be taken into consideration of the inventor of this present invention, that another form of a dual cancer diagnostic kit, could be to identify any probably significant rejection of carcinoma or metastases thereof, theoretically be another combination of cancer related test kit, namely to detect a decreasing level of CI IA during treatment with infusion of for instance recombinant anti human CI IA, which is the focus of this invention - a breakdown of carcinoma or other cancer tissue, evidenced by a decreasing CI IA, and an elevated C4d subunit.
Therefore, a second specific test kit aimed at monitoring cancer cell lysis based on the treatment with infusion of antihuman CI IA antibody, or may be even other form of immune treatment that would cause rejection of cancer cells. Such test kit could according to the inventor be an important tool to monitor possible rejection of cancer, and may be also for monitoring of quite another form of rejection, namely detection of rejection occurring after allogenic organ - or bone marrow transplantation, where most probably, besides the increased activity of the classical complement cascade pathway, For instance indicated by a decreased CI IA and an increased native C4d unit. So, as an alternative cancer diagnostic kit could consist of measurement of CI IA (to identify a possible decrease) held together with an increased
concentration of C4d.
This could actually also be a test kit for measuring threatening rejection of an organ, bone marrow or umbilical cord blood transplantation, and
The relation between utilizing the findings in this invention both as diagnostic and in particular also as a novel treatment module.
The object of the present invention relates to the unique CI IA coating of certain human cancers, especially carcinoma, certain sarcomas and primary malignant brain tumors. These types of malignant tumors represent the most dominant malignant organ related types of tumors, where these cancer types appear to escape the human immune defence system including the
complement system.
The equipment used for the measurement of CI IA on carcinoma cells and malignant brain tumor cells as well as measurement of benign cells all harvested from patients by either biopsies from the tumor or by pleura or ascites effusion from patients with metastatic carcinomas. The cells were cultured on sterile micro-slides in Leighton tubes and a corresponding NUNC culture flask as monolayer cultures in MEMF12 medium at Danish laboratories in 37°C C02 incubators.
The inventor is aware of the complexity of the various forms of complement components and what and how the various parts or factors in the complement system which is activated and in what order. It is for instance the inventor's opinion that it is in all circumstances the so-called "Classical Pathway" of the complement system that is inhibited in the case of cancer cells and their CI IA coat, the part of the complement system suffering or inhibited from being activated at least on the location of the primary cancer and/or the metastases.
The complement system consists of a number of small proteins found in the blood, in general synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex
The complement system is a part of the immune system that helps or complements the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the innate immune system, which is not adaptable and does normally not change over the course of an individual's lifetime.
The complement system is activated when for instance adaptive immune is initiated. There are three pathways through which the complement system can be activated.
1. The "classical pathway" starts at complement component Clqrs
complex, due to an antigen - antibody reaction on a surface of a cell. The initiation of this pathway is regulated and under control by an inhibitor protein, called CI inhibitor or CI inactivator (CI IA). The CI IA also called Cl-inhibitor (Cl-inh, CI esterase inhibitor) is a protease inhibitor belonging to the serpin superfamily. The author of this invention is attempting to call this protease inhibitor for "CI IA". Its
main function is the inhibition of the complement system to prevent spontaneous activation where the ultimate end point of a classical pathway is cell lysis.
The "lectin pathway" is a type of cascade reaction in the complement system, similar in structure to the classical complement pathway proceeding directly through the action of C4 and C2 to produce activated complement proteins further down the cascade. In contrast to the classical complement pathway, the lectin pathway does not recognize an antibody bound to its target. The lectin pathway starts with mannose-binding lectin to certain glycoproteins of certain microorganisms. This pathway consists of lectin binding to mannose, or glucose determinants on glycoprotein components on microorganisms such as salmonella, listeria, and neisseria, fungal pathogens, and certain types of virus such as HIV-1 and RSV.
The "alternative pathway" is one of the three complement pathways that opsonize and kill pathogens. The pathway is triggered by hydrolysis of complement component 3 when the C3b protein directly binds the microbe. As described above the classical pathway of a complement cascade is normally related to an adapt immune reaction such as an antigen-antibody reaction in which IgG or IgM is involved in the antigen-antibody reaction on the surface of a pathological cell or microorganism; an exception for this rule is that IgG4 cannot activate the classical pathway.
The classical pathway appears to be efficiently inhibited on certain cancer cells such as carcinomas, certain types of sarcomas and malignant primary brain tumors such as astrocytomas, glioblastomas, etc. (Osther K, et al.
Demonstration of a complement inactivator on cultured cells from human malignant brain tumors, Acta Neurol. Scandinav.50 :681-689).
The present invention relates to the profound change, the coating of CI IA plays in inhibiting not only the CI qrs activation but also the lectin activation.
By efficiently removing or neutralizing the inhibitory effect of the CI IA on the plasma cell membrane of cancer cells by for instance the antigen-antibody neutralization of CI IA (except for IgG4, which cannot activate the classical pathway).
In particular, it is an object of the present invention to provide a novel combined immunological-complement activating reaction on cancer cells to promote lysis of these cells, and a test kit that possibly can disclose a possible appearance of cancer in patients, consisting of measurement of the level of CI IA and the complement component "native or naive C4" instead of trying to identify C4 subunits. In this manner the cancer test diagnostic kit can be used to detect cancer or metastases from cancer at an earlier stage of the cancer disease in patients, who otherwise get identified late, - often up to one year or more into their cancer disease. Treatment approach using human or humanized IgG antibody against CI IA and in vitro and in vivo diagnostic method and kits for detecting and
monitoring cancer of type carcinoma and malignant brain cancers.
The available methods present now a days to develop and actually produce both diagnostics using the measurement of CI inactivator and naive C4 in blood and may be in spinal fluid, even though it is anticipated that malignant brain tumors - in spite of their more or less exclusive growth in the CNS without metastasizing into the rest of the body, may appear that the CI inactivator released by the malignant brain tumor (primary or secondary), may release the and especially treatment using measurement of the I.V. blood content of CI inactivator and naive C4 in peripheral blood may also reflect tumor growth in the brain.
As an interesting different aspect of measuring the two proteins described is the curiosum that combination of measurement of CI inactivator and C4 level can be utilized in other diagnosis than cancer such as C4 and levels of CI INH and function may be useful to monitor treatment effects of patients with HANE or Quincke's edema, where both blood CI IA and C4 will be extremely low. Therefore, certain types of malignant diseases, such as certain lympho- proliferative diseases such as certain types of lymphoma may in certain
circumstances appear to get allergy-like swellings, some times indistinguishable to Quinckes edema. In these cases the paradox, when compared to carcinomas and primary brain tumors, one may find an extremely low CI IA and an extremely low C4 in the blood. So in more rare type of malignant diseases such as Iymphoproiiferative diseases, that instead of finding high blood levels of CI IA and C4 in cancer, - these lymphoma or lymphoma-like diseases may instead show extremely low values of CI IA and C4 in the blood. The in vitro diagnostic method using measurement of CI inactivator and native (naive) C4 and in vivo diagnostic method using humanized anti CI inactivator coupled to a tracer such as technecium
For the in vitro diagnostic purpose, it is part of this invention to use mouse monoclonal anti human CI inactivator antibody and mouse monoclonal anti human C4 antibody to be used for the production of ELISA kits for the measurement of these components in blood and other body fluids. The mouse monoclonal antibody is anticipated to be made by the method described according Proposal for using Creative BioLab's Hybridoma for Monoclonal Antibody manufacturing method for the production of anti human CI inactivator and anti human naive (native) C4 complement component to be used for the testing systems selected, e.g., such as ELISA technology and systems often used at Doctor's office such as Quick Read from Orion
Diagnostics, which can be modified to measure blood samples at physician's office or other immunological measurement methods that ae inexpensive ( Orion Diagnostica Oy, P.O.Box 83, FI-02101 Espoo, Finland).
An in vivo diagnostic approach would be a humanized anti CI IA bound to a tracer such as for instance a radioactive tracer such as Technitium 99, which then could detect for instance via scanning or other measurements producing a picture of where in the body the CI IA is bound, and where there may be metastases. This cancer kit can also be used in monitoring cancer patients after surgery - as exemplified in one of the two patients described in this
invention, monitor the effect of a cancer treatment whether it is by treating with chemotherapy or an isotope with limited radiation, such as beta rays.
At a hospital in Denmark one patient was tested using semipurified porcine anti CI inactivator labelled with technecium99"1 on a patient with suspected carcinoma essentially using the method described by Javadpour (Javadpour N et al.JAMA. 1981 Jul 3;246(l) :45-9). The Tc99m-labeled semipurified porcine CI inactivator was injected intravenously and the patient scanned using methods described by Javadpour. At that time it was reported that images showed anti CI inactivator appeared to bind on at least one major location in the abdominal aorta, which at that time was interpreted as a possible carcinoma site (according to the binding of the labeled antibody)
A humanized monoclonal biphasic anti human CI inactivator antibody labeled to Technicium99m could be a useful for detection of metastases on images using according to known principles
Targeting Anti human CI inactivator using human recombinant IgG
The novel anti CI IA treatment with repetitive infusions capable of being done with novel human anti CI IA from recombinant human cell lines such as for instance Human Embryonic Kidney cells (HEK) cells and more preferably certain Human HEK cell lines, such as for instance HEK293t or HEK293ts cell lines using a proprietary vector system consisting of pST2 for cloning and pST2-HF3 after cloning which was linearized by Bglll and GcIL restriction enzymes (see figure 3).
This vector, which is a proprietary vector system owned by HumanCell (HumanCell, Naperville, Illinois, USA) and is used to prepare a recombinant human IgG, chosen between any of the subtypes of IgG (IgM antibodies may also be used for this purpose), namely all of those subtype of IgG that can activate the classical pathway of the Complement system, except IgG4, which cannot activate the classical pathway of complement.
The anti CI IA recombinant anti CI IA will carry Fab2 antigen determinants directed against CI IA molecule, and perform binding and activation of the classical pathway for Complement all the way through C9, which will cause "pores" in its binding together with the other cell bound complement component.
The pore itself is created by a poly-active effect of C9 binding to the cancer cell membrane, thus causing lysis of the cell. Also humanized monoclonal anti human CI IA can be utilized provided it through its binding to the cancer cell can activate the classical pathway of complement.
The human anti CI IA recombinant IgG or even the humanized monoclonal anti CI IA can be bound or conjugated to a potent chemotherapy, which then can be led all the way directly to the cancer cells carrying CI IA on their plasma membrane, and/or expressing this protein.
A possible novel approach towards a method of treating primary malignant brain tumors
An extremely unique exception is the primary malignant brain tumors such as astrocytomas, gliomas, glioblastomas, etc., which can be recognized by the early study done by Kurt Osther, and published to demonstrate binding of CI IA to human malignant brain tumors (Osther K, Hojgaard K, and Dybkjaer E, Acta Neurol. Scandinav., 1974, 56:681-689).
Without considering the not so clear cut event at that time, which the inventor later on has arrived at, namely the fact that the primary brain cancer cells such as astrocytomas, gliomas glioblastomas, etc. never mestastasizes outside the Central Nervous System (CNS). According to the author of this invention it can now be understood, why these primary brain cancers cannot spread to the rest of the human body except exclusively only being able to spread in the CNS system.
The observation among others that lead the inventor to this conclusion is that these primary brain tumors needs the unique environment to remain alive and
stay malignant even though there are amble possibilities for these primary brain tumors to spread into the blood system in theory has all the chances to trespass through the blood brain barrier and spill into the human (mammal) body outside the CNS system even though they are coated with CI IA and therefore easily should be able to spread outside the CNS system.
It is therefore postulated by the inventor that the reason why he could subculture carcinoma cells and find that these cells rebuild their Cll IA coat, produced by themselves after the plasma membrane of the carcinoma cells had been stripped from the plasma bound CI IA coat, they could easily reconstruct the CI IA coat when being sub-cultured without any problems.
From the experiments demonstrated in 1976 by the inventor that the primary brain cancer cells, astrocytomas, etc. showed in the primary culturing process a clearcut CI IA coat, but when subcultured as was done with the carcinoma cells, the primary brain cancer cells lost this ability. Biopsies from secondary (metastatic cancer in the brain) consisted of carcinoma metastases to the brain. Carcinoma metastases to the brain - also called secondary or "non primary" brain cancer retained their CI IA coat after having been trypsinized and sub- cultured, exactly as the carcinoma cells cultured from tumor biopsies outside the brain. Therefore, it was obvious that even though primary brain cancer cells are using the same CI IA coating in the primary culture and therefore can block the classical pathway of the complement system, and therefore resist any immune attack, but when entering the human body outside the CNS system, they do not get the same CNS nutrients, proteins, cytokines or whatever differentiate them from the carcinoma cells. Treatment approach using anti human CI IA
The approach to treatment by induced rejection caused by anti CI IA as an Antigen-Antibody reaction that neutralize the CI IA coat on the plasma membrane of cancer cells
According to this invention, it shall be possible to be able to treat a cancer patient with several consecutive injections or infusions with an anti human CI IA antibody of IgG or IgM type (minus IgG4) that ideally shall be tolerated to be repetitive infused or injected ideally for months to years, ideally without immunizing the patient, contrary to what was possible for me to do with a xenogeneic anti CI IA mixed with other antigen determinants, one of these being a protein that I at that time called anti CI IAC (an antibody that later on appeared to be C-reactive protein (CRP), which actually only is an
inflammatory protein, present as an acute phase protein appearing when patients are infected with microorganisms, such as for instance bacteria.
Dosage ranges
A postulated dosage range using specific recombinant human or humanized antihuman CI IA would be starting up with I.V. dosage ranges 1 - 2 gram per kg (such as 1.0 gram per kg, e.g. 1.1 gram per kg, such as 1.2, gram per kg, e.g. 1.3 gram per kg, such as 1.4, gram per kg, e.g. 1.5 gram per kg, such as 1.6, gram per kg, e.g. 1.7 gram per kg, such as 1.8, gram per kg, e.g. 1.9 gram per kg, such as 2.0 gram per kg) given for instance once or twice a month until effect is seen, but depending upon the specificity the doses may be much smaller in size.
10 to 50 gram of anti CI IA human immunoglobulin with a measurable antibody titer determination of the recombinant specific immunoglobulin with concomitant measurement of the concentration of CI IA and C4 in the blood.
Thus, in a particular interesting embodiment of the invention, the dosage form comprises 10-50 gram anti CI IA human immunoglobulin, such 11-49 gram anti CI IA human immunoglobulin, e.g. 12-48 gram anti CI IA human immunoglobulin, such 13-47 gram anti CI IA human immunoglobulin, e.g. 14- 46 gram anti CI IA human immunoglobulin, such 15-45 gram anti CI IA human immunoglobulin, e.g. 16-44 gram anti CI IA human immunoglobulin, such 17-43 gram anti CI IA human immunoglobulin, e.g. 18-42 gram anti CI IA human immunoglobulin, such 19-41 gram anti CI IA human
immunoglobulin, e.g. 20-40 gram anti CI IA human immunoglobulin, such 12- 39 gram anti CI IA human immunoglobulin, e.g. 22-38 gram anti CI IA
human immunoglobulin, such 23-37 gram anti CI IA human immunoglobulin, e.g. 24-36 gram anti CI IA human immunoglobulin, such 25-35 gram anti CI IA human immunoglobulin, e.g. 26-34 gram anti CI IA human
immunoglobulin, such 27-33 gram anti CI IA human immunoglobulin, e.g. 28- 32 gram anti CI IA human immunoglobulin, such 29-31 gram anti CI IA human immunoglobulin.
In a preferred embodiment the anti CI IA human immunoglobulin or the recombinant human or humanized antihuman CI IA is administered 1-5 times a month, such as 2-4 times a months, e.g. 2-4 times a month and in an even more preferred embodiment 1 or 2 times a month.
In a preferred embodiment the anti CI IA human immunoglobulin or the recombinant human or humanized antihuman CI IA is administered weekly or once every second week.
Of course the tumor load is followed by clinical investigation including x-ray, MR Scanning, CT scanning or by using endoscopic methods.
In an embodiment a preferred administration for is IV administration (intra venous administration). Method for the production of CI esterase inhibitor
In one aspect the present invention pertains to a method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines. It may be preferred that the recombinant CI esterase inhibitor is recombinant human CI esterase inhibitor made in human cells and not in non human (mammal cells such as CHO or alike).
In another embodiment, the HEK cell line is HEK 293T cell line (HumanCell, Naperville, Illinois, USA).
5 It may be preferred that the human host cell is cultured to a cell density of above 103 cells per ml.
In an embodiment no synthetic protease inhibitor(s) is/are added.
10 In a further embodiment the said CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
a. the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 b. a subsequence of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2;
15 c. a sequence which has at least 85% sequence identity with any one of the sequences set forth in i) and ii).
In another embodiment, the amino acid comprises a sequence sharing at least 86 % identity with that set forth in SEQ ID NO. : l or SEQ ID NO. : 2 , such as 20 87 % identity, 88 % identity, 89 % identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, or 99 % identity.
In one embodiment the sequences in ii) and iii) are immunologically
25 equivalent to the sequence set forth in i).
In a further embodiment said CI esterase inhibitor has a carbohydrate moiety, which constitutes from 45-50% of its molecular mass.
30 In yet an embodiment said CI esterase inhibitor comprises sialic acid, such as in the same amounts as native CI esterase inhibitor.
In another embodiment, cells are transfected with IgG to which the gene protope to CI IA antigen of the determinants of an IgG said sequences in ii) and iii) are capable of reacting specifically with an antiserum/polyclonal antibody or a monoclonal antibody raised against a polypeptide having the sequence set forth in i).
In an embodiment the monoclonal antibody is a mouse monoclonal antibody selected from the group consisting of: Clone 3F4-1D9 (commercially available from Bio-Rad AbD Serotec, Lifespan Biosciences, Merck Millipore, Abnova and United States Biological) , clone LS-C39048 (commercially available form Lifespan Biosciences ) , clone [8D4C12E6 (commercially available from Sino Biological), clone 350507 (commercially available from R&D Systems), clone abxl0017 (commercially available from Abbexa), clone KT28 (commercially available from Thermo Fisher Scientific, Inc. and Abbiotec), clones 8F3 and 6C6 (commercially available from Abbiotec), clone 10K343 (commercially available from United States Biological), clone GWB-2F0410 (commercially available from Genway), clone 119-15582 (commercially available from Raybiotech), clone CAB-4608MH (commercially available from Creative Biomart), clone Bll (commercially available from Santa Cruz Biotechnology), clone MAA235Hu22 (commercially available from Cloud-Clone Corp), clones EPR8016, M81, EPR8015, ab54898 (commercially available from Abeam).
In yet an embodiment the CI esterase inhibitor which comprises an amino acid sequence, which is a subsequence of the sequence set forth in SEQ ID NO. : 1, has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or the sequence set forth in SEQ ID NO. : 2.
In an embodiment said CI esterase inhibitor comprising a sequence, which has at least 85% identity to the sequences set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 or to a subsequence thereof, has an inhibitory effect on the
complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
In a further embodiment the inhibitory effect of said CI esterase inhibitors is measured in an in vitro assay comprising the steps of:
i) incubating from 2.5 ng to 1000 ng of the CI esterase inhibitors with 1 ng of factor Xlla in 20 μΙ of 50 mM Tris-HCI buffer at 37°C for 5 minutes;
ii) adding 1 pg purified prekallikrein (in 10 pi Tris-HCI) followed by incubation at 37°C for 1 minute;
ii) adding 20 μΙ Tris-HCI and 50 pi s-2302 chromogenic substrate for plasma kallikrein, FXIa and FXIIa (H-D-Pro-Phe-Arg-p-nitroaniline [pNA] -2HCI, DiaPharma Group, Inc., West Chester, Ohio), followed by incubation at 37°C for 10 minutes and addition of 50 μΙ acetic acid to stop reaction; iii) determining kallikrein activity by reading the OD at 405 nm.
In yet an embodiment said CI esterase inhibitor consists of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
In a further embodiment said exogenous nucleic acid sequence comprises a sequence selected from the group consisting of
i) SEQ ID NO. : 3 or SEQ ID NO. : 4 (NCBI Reference Sequence:
NM_000062.2)
ii) a subsequence of the sequence defined in i);
iii) a sequence which has at least 85% nucleic acid identity with the sequence set forth in i) or ii).
In another embodiment, the nucleic acid comprises a sequence sharing at least 86 % identity with that set forth in SEQ ID NO. : 3 or SEQ ID NO. :4 , such
as 87 % identity, 88 % identity, 89 % identity, 90 % identity, 91 % identity, 92 % identity, 93 % identity, 94 % identity, 95 % identity, 96 % identity, 97 % identity, 98 % identity, or 99 % identity. In an embodiment said exogenous nucleic acid sequence encoding said CI esterase inhibitor has been inserted into a plasmid vector
consisting/substantially consisting of the sequence set forth in SEQ ID NO. : 5 (Hzec6). Sequence identity
As commonly defined "identity" is here defined as sequence identity between genes or proteins at the nucleotide or amino acid level, respectively.
Thus, in the present context "sequence identity" is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100).
In one embodiment the two sequences are the same length.
In another embodiment the two sequences are of different length and gaps are seen as different positions.
One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilised. Alternatively, PSI-Blast may be used to perform an iterated search which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.
The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation.
Production of antibodies
In an embodiment the invention pertains to a method to the production of monoclonal antibodies against CI IA and native C4
In a further embodiment the invention pertains to the production of
humanized monoclonal antibody against CI IA
In another embodiment, the invention pertains to a method for producing recombinant human IgG and subtypes of IgG except IgG4 by transfecting HEK cells using a vector capable of containing the antigenic determinants against CI IA, inducing classical pathway reaction of complement.
In another aspect the invention also pertains to the production of recombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI IA developed from the anti CI IA fab. In another aspect the invention also pertains to the production of
recombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI IA developed from the anti CI IA fab
Human host cell
In a further aspect the invention pertains to a human host cell selected from the group consisting of an immortalized cell from the placenta, an
immortalized cell from the human cells such as HEK cells, PerC6 cells, amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a CI esterase inhibitor.
In one embodiment the human host cell comprises an exogenous nucleic acid sequence as defined as the Coding region also defined as SEQ ID NO: 3 cloned into pST2 expression vector In a further embodiment the human host cell expresses a CI esterase inhibitor as defined above.
It may be preferred that the human host cell is 24. The human host cell according to any one of claims 20-23, wherein said host cell is a HEK 293 cell.
Recombinant CI esterase inhibitor
In another aspect the invention pertains to a recombinant CI esterase inhibitor obtainable by the method above. Compositions
In a further aspect the invention pertains to a composition comprising a recombinant CI esterase inhibitor, characterised in that
i) said composition comprises substantially no synthetic protease inhibitors, and/or
ii) said composition is serum free.
It may be preferred that the recombinant CI esterase inhibitor is as defined as above. Use of recombinant CI esterase inhibitor and administration
In a further aspect the present invention pertains to a recombinant CI esterase inhibitor or a composition comprising recombinant CI esterase inhibitor for use as a medicament. In an embodiment the recombinant CI esterase inhibitor or a composition comprising recombinant CI esterase inhibitor may be used for the preparation
of a medicament for the treatment and/or prevention and/or reduction of ischemia.
In a further embodiment the recombinant CI esterase inhibitor or a
composition comprising recombinant CI esterase inhibitor may be used for the preparation of a medicament for the treatment and/or prevention and/or reduction of Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation). In another embodiment the recombinant CI esterase inhibitor or a
composition recombinant CI esterase inhibitor may be used for the
preparation of a medicament for the treatment and/or prevention and/or reduction of Inflammation in spinal cord injury. In yet an embodiment the recombinant CI esterase inhibitor or a composition recombinant CI esterase inhibitor may be used, for the preparation of a medicament for the treatment of burn wounds (promotion of re- epithelialization, prevention/alleviation of granulation tissue development and scar formation).
In a further embodiment the recombinant CI esterase inhibitor or a
composition recombinant CI esterase inhibitor may be used, for the
preparation of a medicament for the treatment and/or prevention and/or reduction of Hereditary angioedema/ Quincke's edema.
In another embodiment the CI esterase inhibitor or composition may be administered intracerebral^, intraspinally, intrathecal^, or intravenously.
In another aspect the present invention pertains to a method for preventing, treating and/or alleviating Ischemic stroke (reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI
esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor.
In another aspect the present invention pertains to a method for preventing, treating and/or alleviating Inflammation in spinal cord injury comprising administering to a subject in need thereof a recombinant CI esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor.
In another aspect the present invention pertains to a method for treating burn wounds (Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor, or a composition comprising recombinant CI esterase inhibitor. Said CI esterase inhibitor or composition comprising recombinant CI esterase inhibitor may be administered intracerebral^, intraspinally, intrathecally, or intravenously
Anti-Cl esterase inhibitor
In another aspect the invention pertains to a an antibody raised against a CI esterase inhibitor according to a method using recombinant human anti human CI inactivator IgG using cloned IgGl cloned into pST2 expression vector In an embodiment the antibody is a monoclonal antibody.
In an embodiment the antibody is a humanized monoclonal antibody
In a further embodiment the antibody is a polyclonal antibody/antiserum.
In a further embodiment the antibody may be a polyclonal antibody raised against a composition comprising semi-purified CI esterase inhibitor.
In another embodiment the said composition may comprise one or more components selected from the group consisting of orosomucoid, alpha2 HS
glycoprotein and Zn alpha2 glycoprotein, C-reactive protein.
In yet an embodiment the antibody may be conjugated to a detectable marker.
It may be preferred that the marker is a fluorescent marker. In another embodiment the marker may be a fluorescein isothiocyanate derivative.
In a further embodiment the antibody may be conjugated to a
chemotherapeutic drug.
In an embodiment the chemotherapeutic drug is selected form the group of chemotherapeutic drugs that hit the cancer cells in the M or S phase and not the GO (resting phase), ,cell killing drugs such as Adriamycin, 5 FU.
Daunomycin, Cis Platinum, etc.
In an embodiment the chemotherapeutic drug is be selected form the group consisting of chemotherapy drugs, alkylating agents and platinium drugs. In a further embodiment the alkylating agents is selected from the group consisting of nitrogen mustards, such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan, nitrosoureas such as streptozocin, carmustine (BCNU), and lomustine, alkyl sulfonates such as busulfan, triazines such as dacarbazine (DTIC) and temozolomide (Temodar®), ethylenimines such as thiotepa and altretamine (hexamethylmelamine).
In a further embodiment the platinum drug is selected from the group consisting of cisplatin, carboplatin and oxalaplatin. are sometimes grouped with alkylating agents because they kill cells in a similar way. Antimetabolites interfere with DNA and RNA growth by substituting for the normal building blocks of RNA and DNA. These agents damage cells during the S phase, when the cell's chromosomes are being copied. They are commonly used to treat
leukemias, cancers of the breast, ovary, and the intestinal tract, as well as other types of cancer.
Examples of antimetabolites include: · 5-fluorouracil (5-FU)
• 6-mercaptopurine (6-MP)
• Capecitabine (Xeloda®)
• Cytarabine (Ara-C®)
• Floxuridine
· Fludarabine
• Gemcitabine (Gemzar®)
• Hydroxyurea
• Methotrexate
• Pemetrexed (Alimta®)
In another embodiment the antibody may be used as a medicament.
In another aspect the invention pertains to an antibody raised against a CI esterase inhibitor, wherein the CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
a. the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 b. a subsequence of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2;
c. a sequence which has at least 85% sequence identity with any one of the sequences set forth in i) and ii),
In an embodiment the antibody may be for use in the manufacture of a medicament for the treatment of a malignant carcinoma, squamous
carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon
carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney
carcinomas bladder carcinomas, malignant brain tumors (e.g. glioblastomas or astrocytomas), astrocytoma, glioblastomas grade III-IV, gliomas, glioblastoma multiforme, oligodendrogliomas, ependymoma, and medulloblastoma. In another aspect the present invention pertains to a method for treating a malignant carcinoma including malignant brain tumors (e.g. glioblastomas or astrocytomas), said method comprising administering to a subject in need thereof an antibody. In another embodiment the antibody may be used in the manufacture of a medicament for treatment of a malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas. The cancer may be a malignant brain tumor such as but not limited to
Astrocytoma or glioblastomas grade III-IV, gliomas, glioblastoma multiforme, oligodendrogliomas, ependymoma and medulloblastoma.
A method for detecting and/or screening and/or monitoring cancer in an individual
One aspect of the present invention provides a method for determining and/or screening cancer in an individual suitable to facilitate the early diagnosis of a cancer. It is another aspect of the present invention to provide a method for monitoring the recurrence of a cancer, status of a cancer or the effect of cancer treatment in an individual.
It is a third aspect of the present invention to provide a kit or device for performing the method according to the invention, having a simple and inexpensive design, being quick and easy to use and not requiring the assistance of specialists or the use of specialised equipment.
The terms excreta and sample are used herein interchangeably.
This is achieved according to the invention as it has surprisingly been found by the inventors that high levels of CI esterase inactivator (CI IA) in combination with low levels of complement component native C4 in human excreta is indicative of cancer.
It is therefore possible according to the present invention to provide an easy and inexpensive method for early detection and monitoring of a cancer in an individual or large populations using the method of the present invention.
In order not to obtain any false-positive results it is important to determine a discriminating value, which divides the tested individuals in a group having either a high or low likelihood of having cancer.
The discriminating value is established by measuring the total concentration of CI IA in both a healthy control population and a population with known cancer and thereby determining the discriminating value. The discriminating value identifies the cancer population with either a predetermined specificity or a predetermined sensitivity or both, and is based on an analysis of the relation between the concentration values and the known clinical data of the healthy control population and the cancer patient population.
The discriminating value is established by measuring the total concentration of C4 in both a healthy control population and a population with known cancer and thereby determining the discriminating value. The discriminating value identifies the cancer population with either a predetermined specificity or a predetermined sensitivity or both, and is based on an analysis of the relation
between the concentration values and the known clinical data of the healthy control population and the cancer patient population.
The discriminating value determined in this manner is valid for the same experimental set-up in future individual tests.
Specificity and sensitivity
The sensitivity of any given diagnostic test define the proportion of individuals with a positive response who are correctly identified or diagnosed by the test, e.g. the sensitivity is 100%, if all individuals with a given condition have a positive test. The specificity of a given screening test reflects the proportion of individuals without the condition who are correctly identified or diagnosed by the test, e.g. 100 % specificity is, if all individuals without the condition have a negative test result.
Sensitivity is defined as the proportion of individuals with a given condition (e.g. cancer), who are correctly identified by the described methods of the invention (e.g. has a positive test-result). Specificity herein is defined as the proportion of individuals without the condition (e.g. cancer), who are correctly identified by the described methods of the invention (e.g. has a negative test result)
Receiver-operating characteristics
Accuracy of a diagnostic test is best described by its receiver-operating characteristics (ROC) (see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577). The ROC graph is a plot of all of the
sensitivity/specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed.
The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly
distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease, latent or recent infection versus no infection, or benign versus malignant disease. In each case, the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results] . This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x axis is the false-positive fraction, or 1 - specificity [defined as (number of false- positive results) / (number of true- negative + number of false-positive results)] . It is an index of specificity and is calculated entirely from the unaffected subgroup.
Because the true-and false-positive fractions are calculated entirely
separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/- specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false- positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. (If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa.) Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.
One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global
measure is the area under the ROC plot. By convention, this area is always > 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0). Thus, it is an object of preferred embodiments of the present invention to provide a method for diagnosing cancer in an individual, the method comprising : a) determining the level of CI IA and native C4 in a sample of said individual, b) constructing a percentile plot of the CI IA and native C4 levels obtained from a healthy population c) constructing a ROC (receiver operating characteristics) curve based on the CI IA and native C4 levels determined in the healthy population and on the CI IA and native C4 levels determined in a population with known cancer d) selecting a desired specificity e) determining from the ROC curve the sensitivity corresponding to the desired specificity f) determining from the percentile plot the CI IA and native C4 levels corresponding to the determined sensitivity; and
g) predicting the individual to have cancer, if the level of CI IA in the sample is equal to or higher than said CI IA level corresponding to the determined specificity and if the level of native C4 in the sample is lower than said C4 level corresponding to the determined specificity, and h) predicting the individual as unlikely or not to have cancer if the level of CI IA in the sample is lower than said total CI IA level corresponding to the determined specificity and if the level of native C4 in the sample is equal to or above than said total C4 level corresponding to the determined specificity.
Thus, it is another object of preferred embodiments of the present invention to provide a method for diagnosing cancer in an individual, the method
comprising : a) determining the level of CI IA and native C4 in a sample of said individual, b) constructing a percentile plot of the CI IA and native C4 levels obtained from a healthy population c) constructing a ROC (receiver operating characteristics) curve based on the CI IA and native C4 levels determined in the healthy population and on the CI IA and C4 levels determined in a population with known cancer d) selecting a desired sensitivity e) determining from the ROC curve the specificity corresponding to the desired sensitivity
f) determining from the percentile plot the CI IA and C4 levels corresponding to the determined sensitivity; and g) predicting the individual to have cancer, if the level of CI IA in the 5 sample is equal to or higher than said CI IA level corresponding to the determined sensitivity and if the level of native C4 in the sample is lower than said C4 level corresponding to the determined sencitivity, and
10 h) predicting the individual as unlikely or not to have cancer if the level of CI IA in the sample is lower than said total CI IA level corresponding to the determined sensitivity and if the level of native C4 in the sample is equal to or above than said total C4 level corresponding to the determined sensitivity.
15
The specificity of the method according to the present invention may be from 70% to 100%, more preferably 80% to 100%, more preferably 90% to 100%. Thus in one embodiment of the present invention the specificity of the invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 20 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
The sensitivity of the method according to the present invention may be from 70% to 100%, more preferably 80% to 100%, more preferably 90% to 100%. Thus in one embodiment of the present invention the sensitivity of the
25 invention is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
The levels of CI IA and native C4 are compared to a set of reference data or a reference value such as the cut-off value to determine whether the subject is 30 at an increased risk or likelihood of e.g. cancer.
To determine whether the patient is at increased risk of developing e.g.
infection, a cut-off must be established. This cut-off may be established by the laboratory, the physician or on a case by case basis by each patient. Alternatively cut point can be determined as the mean, median or geometric mean of the negative control group ((e.g. healthy population, healthy individual, population with known cancer or individual with known cancer) +/- one or more standard deviations or a value derived from the standard deviation)
Risk assessment
The present inventors have successfully identified a new marker for measuring a cell-mediated response to an antigen. The concentration of the marker IP-10 is increased in subjects with a cell mediated immune-response to an antigen. And IP-10 appears to be an efficient marker for detection of e.g. infection with M. tuberculosis.
The sensitivity is higher than any other established marker and the specificity is comparable or better than with any other established marker, see e.g.
example 4, 5 and 10).
Statistical reasoning can for example be based on the risk of having the disease depending on age, occupation, exposure, genetic background, HLA- type.
Cut-off points can vary based on specific conditions of the individual tested such as but not limited to the risk of having the disease, occupation, geographic residence or exposure. Cut-off points can vary based on specific conditions of the individual tested such as but not limited to age, sex, genetic background (i.e. HLA-type), acquired or inherited compromised immune function (e.g. HIV infection, diabetes, patients with renal or liver failure, patients in treatment with
immune-modifying drugs such as but not limited to corticosteroids,
chemotherapy, TNF-a blockers, mitosis inhibitors).
Doing adjustment of decision or cut-off limit will thus determine the test sensitivity for detecting an infection, if present, or its specificity for excluding infection or disease if below this limit. Then the principle is that a value above the cut-off point indicates an increased risk and a value below the cut-off point indicates a reduced risk. In addition test samples with indeterminate results must be interpreted separately. Indeterminate results are defined as result with an unexpectedly low level of IP-10 in the mitogen stimulated sample (PHA). The final cut point for an indeterminate IP-10 result may be decided according to the study group, especially in immunosuppressed the cut-off level may be selected at a lower level.
Cut-off levels
As will be generally understood by those of skill in the art, methods for screening/monitoring/determining cancer are processes of decision making by comparison. For any decision-making process, reference-values based on subjects having the disease and/or subjects not having the disease, infection, or condition of interest are needed.
The cut-off level (or the cut-off point) can be based on several criteria including the number of subjects who would go on for further invasive diagnostic testing, the average risk of having and/or developing e.g. cancer to all the subjects who go on for further diagnostic testing, a decision that any subject whose patient specific risk is greater than a certain risk level should go on for further invasive diagnostic testing or other criteria known to those skilled in the art.
The cut-off level can be adjusted based on several criteria such as but not restricted to certain group of individuals tested. E.g. the cut-off level could be
set lower in individuals with known cancer, cut-off may be higher in groups of otherwise healthy individuals with low risk of developing cancer.
In one embodiment the present invention discloses a method for determining if a subject is likely of having cancer, which comprises:
(a) obtaining from the subject a sample, and
(b) quantitatively determining the concentration of CI IA and native C4 present in the sample, the presence of the CI IA polypeptide present in the sample at a concentration equal to or higher than the selected and the presence of the native C4 polypeptide present in the sample at a concentration lower than the selected cut-off, indicating the that the subject is likely to have cancer.
More specifically one aspect of the present invention relates to a method of diagnosing cancer, comprising the steps of a) incubating a sample obtained from an individual with anti-Cl IA and incubating another sample obtained from the same individual with anti-
C4 b) determining the Cl-IA and C4 level in said samples c) comparing said determined CI IA and C4 level with reference levels, thereby determining whether the individual is likely of having cancer.
The discriminating value is a value which has been determined by measuring the parameter or parameters in both a healthy control population and a population with known cancer thereby determining the discriminating value which identifies the cancer population with either a predetermined specificity or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control
population and the cancer patient population, such as it is apparent from the detailed discussion in the examples herein. The discriminating value
determined in this manner is valid for the same experimental setup in future individual tests.
In the specific experimental setups described herein (example 1, figure 1), the level threshold of CI IA useful as a cut-off value was found to be in the range of but not limited to 40 mg/100 ml sample. Preferably the cut-off is in the range between 15 - 40 mg/100 ml. Normal serum concentrations of CI IA is in the range from 15-40 mg/ml serum whereas normal serum concentrations of native C4 is in the range from 15-50 mg/lOOml.
Preferably the cut-off value indicating a suspected level of CI IA would be 45 mg/100 ml (and a level of 60 mg/100 ml of native C4, see below).
In the specific experimental setups described herein (example 1), the level threshold of native C4 useful as a cut-off value was found to be in the range of but not limited to 20 mg/100 ml to 50 mg/100 ml. Preferably the cut-off is in the range between 20 mg/100 ml and 60 mg/100 ml
Preferably the cut-off value is over 60 mg/100 ml
Thus, based on the above, if a patent has a CI IA level at or above the cut-off level for CI IA and a native C4 level below the cut-off level for C4 the patent is likely to have cancer.
The multivariate DISCRIMINANT analysis and other risk assessments can be performed on the commercially available computer program statistical package Statistical Analysis System (manufactured and sold by SAS Institute Inc.) or by other methods of multivariate statistical analysis or other statistical software packages or screening software known to those skilled in the art.
As obvious to one skilled in the art, in any of the embodiments discussed above, changing the risk cut-off level of a positive test or using different a priori risks which may apply to different subgroups in the population, could change the results of the discriminant analysis for each patient.
In yet an embodiment of the present invention, the amount of CI IA and native C4 may be measured by a device, said device is selected from the group consisting of an assay, an immunoassay, a stick, a dry-stick, an electrical device, an electrode, a reader (spectrophotometric readers, IR- readers, isotopic readers and similar readers), histochemistry, and similar means incorporating a reference, filter paper, colour reaction visible by the naked eye.
The method according to the invention can equally well be used for monitoring the response to treatment and the progress of the cancer as rising CI IA values in combination with decreasing C4 values may suggest that a patient could have a negative development of the cancer. In such cases the
discriminating value is set specifically for each individual. The cancer may be selected from the group consisting of... malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney carcinomas bladder carcinomas, ; including malignant brain tumors (e.g. glioblastomas or astrocytomas).... Astrocytoma or glioblastomas grade III-IV, gliomas, glioblastoma multiforme,
oligodendrogliomas, ependymoma, medulloblastoma.
The method according to the invention may be used both for an individual and for an entire population, but more appropriately to a population already identified as having an increased risk of developing cancer, e.g. individuals with a genetic disposition, individuals who have been exposed to carcinogenic substances, or individuals with cancer-predisposing non-malignant diseases.
When an individual has been identified as having high CI IA levels in combination with low C4 in his or her excreta, the individual should be referred for further examination. If a cancer is found, the patient could be offered surgery, radiation or adjuvant anti-neoplastic therapy aiming at curing the patient of cancer.
Prognosis
In one embodiment, CI IA and native C4 may be used for predicting the prognosis of subjects diagnosed with cancer. When used in patient prognosis the method according to the present invention may help to predict the course and probable outcome of the cancer, thus assisting the skilled artisan in selecting the appropriate treatment method and predict the effect of a certain treatment for the condition.
Monitoring
In one embodiment, CI IA and native C4 may be used for monitoring subjects diagnosed with cancer. When used in patient monitoring the method according to the present invention may help to assess efficacy of treatment during and after termination of treatment e.g. monitoring and predicting possible recurrence of the cancer.
The possibility to monitor therapy efficacy by the present invention is particularly relevant because:
a) it is easy to perform by a simple blood draw instead of currently available methods like regular cat scans, MR scans, other expensive x- ray methods, and explorative surgery for occult cancer,
b) by using the relatively inexpensive measurement of CI IA and native C4 it may be possible to identify cancer in patients before they have significant symptoms of cancer. The testing could conveniently be done at clinics housing several physicians, or referral laboratories, at hospital laboratories and as follow up on laboratories serving oncology
departments, malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney
carcinomas bladder carcinomas, ; including malignant brain tumors (e.g. glioblastomas or astrocytomas)....! Astrocytoma or glioblastomas grade III-IV, gliomas, glioblastoma multiforme, oligodendrogliomas, ependymoma, medulloblastoma.
Screening
In one embodiment, the method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of cancer by measuring the level of CI IA and native C4 according to the invention and correlating the levels measured to a pre-specified levels, indicating the presence or absence of cancer.
CI IA and C4A level determination
The levels of CI IA and native C4 or other biological markers is measured by conventional analytical methods, such as immunological methods known to the art.
Measurements of biological markers such as CI IA and C4 can be combined with measurements of other molecules at gene, RNA, or protein level in accordance with the teachings herein.
As stated above, detection of biological markers such as CI IA and C4 may be made at the protein or nucleic acid levels. Consequently, reference to the presence or level of said CI IA, C4 and other relevant biological markers includes direct and indirect data. For example, high levels of CI IA and native C4 mRNA are indirect data showing increased levels of CI IA and C4 respectively. Ligands to CI IA and C4 are particularly useful in detecting and/or quantitating these molecules.
Antibodies to biological markers such as CI IA and native C4 are particularly useful. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays, xMAP multiplexing, Luminex, ELISA and ELISpot. Reference to antibodies includes parts of antibodies, "mammalianized" (e.g. humanized) antibodies, polyclonal, recombinant or synthetic antibodies and hybrid and single chain antibodies.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the biological marker to be measured e.g. CI IA and native C4 or antigenic fragments thereof and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art.
Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the biological marker (e.g. CI IA and C4), or antigenic part thereof, collecting serum or plasma from the animal and isolating specific sera by any of the known immuno-adsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the
homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and
lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
Detection can also be obtained by either direct measure of the biological marker (e.g. CI IA and C4) using specific antibody in a competitive
fluorescent polarization immunoassay (CFIPA) or by detection of
homodimerization of interferon-gamma by dimerization induced fluorescence polarization (DIFP). In either case, detection and quantitation will be down to or less than 6 pg/ml.
Several techniques are known to the skilled addressee for determination of biological markers such as CI IA and native C4. The presence or level of immune effecter may be determined by a Sandwich ELISA method, where the Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form, or variation of that method most recently described for the use of measuring complement components (Kotimaa JP et al. J Immunol Methods. 2015 Apr;419 : 25-34). Luminex beads method from Bio- Rad, called Luminex xMAP technology, Bio-Plex Pro™ magnetic multiplex assays provide researchers the most relevant multiplexing solutions.
Luminex, ELISPOT, mRNA based techniques like RT-PCR or Intracellular flow cytometri.
In order to idenetify patients, who could be used as donors to identify human anti CI inactivator, ELISPOT, could be a convenient method as a screening method for screening Quincke edema, lymphoproliferative or lymphoma patients for autoimmune antibodies against CI inactivator, ELISPOT would be a suitable method, as it was developed by Cecil Czerkinsky's group in
Gothenburg, Sweden in 1983,m for the purpose of detecting antigen-specific Antibody Secreting Cells (ASC) in a B cell ELISPOT assay (Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A (1983). "A solid-phase
enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells". J Immunol Methods 65 (1-2) : 109-121).
Sandwich ELISA method could be convenient method for testing the level of CI inactivator and native C4, as described by Kotimaa et al used on complement components as such (Kotimaa JP et al. J Immunol Methods. 2015 Apr;419 : 25-34) because the test system only have to be active in the mg area, and more difficult to be used in other areas where one is looking for microgram amount. Sample
In one embodiment the sample is derived from the group consisting of blood, serum or spinal fluid (if brain cancer is suspected)
In a preferred embodiment the sample is derived from blood.
Conveniently, when the sample is whole blood, the blood collection tube is treated with anticoagulant (e.g. heparin, or ACD, etc). Notwithstanding that whole blood is the preferred and most convenient sample, the present invention extends to other blood derived sample such as pleural or ascites fluids, and spinal fluid sample.
In one embodiment the present invention thus relates to a method, wherein the sample is derived from blood, urine, pleural fluid, bronchial fluid, oral washings, tissue biopsies, ascites liquid, pus, cerebrospinal fluid, aspirate, and/or follicular fluid.
Generally, blood is maintained in the presence of an anticoagulant (preferably heparin, alternatively e.g. citrate or EDTA). The anticoagulant is present in the blood collection tube when blood is added. The use of blood collection tubes is preferably but not necessarily compatible with standard automated laboratory systems and these are amenable to analysis in large-scale and random access sampling. Blood collection tubes also minimize handling costs and reduce laboratory exposure to whole blood and plasma and, hence, reduce the risk of
laboratory personnel from contracting a pathogenic agent such as but not limited to human immunodeficiency virus.
Alliquots of whole blood may be in volumes ranging from 10μί-4000 μΙ, such as but not limited to 50μΙ_, 100 μΙ, 200 μΙ, 300 μΙ, 400 μΙ, 500 μΙ, 600 μΙ, 700 μΙ, 800 μΙ, 900μΙ, 1000 μΙ, 1100 μΙ, 1200 μΙ, 1300 μΙ, 1400 μΙ, 1500 μΙ, 1600 μΙ, 1700 μΙ, 1800 μΙ, 1900μΙ, 2000μΙ, 2100 μΙ, 2200 μΙ, 2300 μΙ, 2400 μΙ, 2500 μΙ, 2600 μΙ, 2700 μΙ, 2800 μΙ, 2900μΙ or 3000μΙ . Kit
The invention further relates to a kit for measurement of the concentration of CI IA and native C4 in excreta . Such kit may consist of a dipstick for determining CI IA and native C4 in excreta however, other options may be but is not limited to Activity Assay (such as zymography), immunologic assays or a Colour Reaction kit.
The present invention further contemplates a kit for performing the method of the present invention. The kit is conveniently in compartmental form with one or more compartments adapted to receive a sample from a subject such as whole blood, serum, purified cells, biopsies or other material . That
compartment or another compartment may also be adapted to contain heparin where the sample is whole blood .
Generally, the kit is in a form which is packaged for sale with a set of instructions. The instructions would generally be in the form of a method of the present invention - i.e. for diagnosing cancer in a subject.
In one embodiment the kit contains antigen and monoclonal or polyclonal antibodies against Cl-IA and C4, which is specifically reacting with CI IA and C4 respectivly in an immune-assay, or specific binding fragments of said antibodies for use as a diagnostic reagent.
The contemplated kit of the present invention may be in a multicomponent form wherein a first component comprises a multiplicity of blood collection tubes, a second component comprises an antibody-based detection for CI IA, third component comprises an antibody-based detection means for C4, a fourth component comprises a set of instructions.
The assay may also be automated or semi-automated and the automated aspects may be controlled by computer software. The assay of the present invention may be automated or semi-automated for high throughput screening or for screening for a number of immune effecters from the one subject. The automation is conveniently controlled by computer software. The present invention contemplates a computer program product, therefore, for assessing the presence or absence or the level of CI IA and C4, said product comprises:
(1) code that receives, as input values, the identity of a reporter molecule associated with a labelled antibody or mRNA (2) code that compares said input values with reference values to determine the level of reporter molecules and/or the identity of the molecule to which the reporter molecule is attached; and
(3) a computer readable medium that stores the codes.
Still another aspect of the present invention extends to a computer for assessing the presence or absence or level of CI IA and C4, said computer comprises: (1) a machine-readable data storage medium composing a data storage material encoded with machine- readable data, wherein said machine- readable data I comprise input values which identify a reporter molecule associated with a labelled antibody or mRNA;
(2) a working memory for storing instructions for processing said machine- readable data, (3) a central-processing unit coupled to said working memory and to said machine readable data storage medium, for processing said machine readable data to compare said values to provide an
assessment of the identity or level of reporter molecules or of molecules to which they are attached; and
(4) an output hardware coupled to said central processing unit, for receiving the results of the comparison.
Diagnostic method
In a further aspect the invention pertains to a method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
c) a first parameter represented by the concentration of CI esterase
inactivator (CI IA) in at least one excreta from the individual and d) a second parameter represented by the concentration of complement component native C4 (C4) in at least one excreta from the individual wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
In an embodiment the first parameter is the total concentration of CI IA.
In a further embodiment the second parameter is the total concentration of native C4.
In yet an embodiment the second parameter is the combination of the concentration of total native C4
In another embodiment the at least one excreta is selected from the group consisting of blood, serum, saliva, spinal fluid, cerebro spinal fluid, pleura fluid, ascites fluid and urine.
In a further embodiment the concentration of CI IA and/or the concentration of native C4 is obtained any time before operation.
In yet an embodiment the concentration of CI IA and/or the concentration of native C4 is obtained any time after an operation such as 2 weeks post- operation, 1 month post-operation, 1.5 month post-operation, 2 months post- operation, 3 month post-operation, 4 months post-operation, 5 month post- operation, 6 months post-operation, 7 month post-operation, 8 months post operation.
In a further embodiment the combination is performed by logistic regression analysis.
In an embodiment the first discrimination value is determined by determining the total concentration of CI IA in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity. In a further embodiment the second discrimination value is determined by determining the total concentration of C4 in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
In a further embodiment the determination of the concentration is performed by means of an immunoassay or an active assay.
In a further embodiment the immunoassay is an ELISA.
In another embodiment the active assay is zymography. In a further embodiment the cancer is selected from the group consisting of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar
carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas. The method may be used for detection of early stage cancer or for detection of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma);
adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar
carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
The method may also be used for monitoring the response to cancer
treatment.
The method may also be used for monitoring the recurrence of a cancer.
In another aspect the invention pertains to a kit for performing the method above, wherein said kit comprises CI IA and native C4.
In a further aspect the invention pertains to a method of diagnosing rejection of transplants, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
b) incubating excreta from a human with anti-C4 and determining the
native C4 level in said sample
c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4 level with a second reference-level e) determining wither said human is likely of having cancer if the level of Cl-IA is at or above the first reference- level and if the level of native C4 is below the second reference-level.
In a further aspect the invention pertains to a method of diagnosing cancer, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample,
b) incubating a blood sample or exsudate or transudate in connection with a suspected rejection of (transplanted) tissue from a human with anti- C4d and determining the C4dlevel in said sample,
c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4d level with a second reference-level, e) determining whether said human is likely of experience a tissue or bone marrow rejection if the level of Cl-IA is normal or lower than 15 mg/100 ml and if the level of C4d is higher than 50 mg/100 ml.
General
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non- limiting examples.
Description of the figures
Figure 1 discloses the concentration of CI IA and complement component C4 in blood is visualized in three rows for three categories of patients, measured using Laurell Immunoelectrophoresis. Normal serum concentration range of CI IA is 15 - 40 mg /100 ml. Normal serum concentration range for C4 is 15 - 50 mg/ 100 ml. In one case native C4 in blood was as low as 4 mg/100 ml Healthy subjects consisting of blood donors and patients suffering from non- malignant disease had values for both CI IA and C4 in the same range, whereas cancer patients had in major cases higher concentrations of both CI IA and native C4.
Figure 2 discloses a simplified drawing of the complement system, targeting the classical pathway of a complement reaction, which is caused by an antigen-antibody reaction, and during chain of reactions occurring through the pathway until C9, the individual complement components are split up after activation to be bound to the pathogen, e.g., the cell, to perform opsonisation, meaning enhancing phagocytosis of antigen, chemotaxis which is attracting macrophages and neutrophils, ending up after sequential binding of part of the split products of C4 to C9, where C9 consists of several fractions that results in a binding of part of the C4, C2, C3, C5 up to C9, catalyzing the enzymatic degradation of covalently bound complement proteins actually ending up with a perforation of the plasma membrane described as "pores" in the plasma membrane by multiple part of the enzymatic molecule C9, resulting in cell lysis or cell death, in many eventually ending up with agglutination and further phagocytosis.
Figure 3 discloses the pST2 vector for cloning. pST2-HF3 after cloning. pST2- HF3 vector was linearized by Bglll and Pcil restrictions enzymes. A HEK cell line adapted to a suspension cell culture expanded in serum-free medium. The cDNA coding region of Clinactivator is cloned into pST2 expression vector (HumanCell Co. Proprietary) at Srfl site to contruct pST-HF3. After pST2-HF3 construction CI inactivator coding region will be sequenced and confirmed.
The cell line is transfected. The cells are then selected and cells appearing to be good producers will be pooled and transferred, plated and after ~ 7 days, and adapted as described to suspension in serum-free medium. The cells are then grown in shaker flasks in a 37°C C02 humidified incubator. The cell morphology of the suspension culture is then compared to the monolayer culture. Supernatant is tested for the presence of the protein targeted, - in this case the CI inactivator.
The same system is used when producing recombinant IgGi , e.g., after the study and evaluation of the paratope for the antigenic determinant (e.g., human CI inactivator), and have been annealed to the antigen determinant in the F(ab')2 "Y" end of the recombinant immunoglobulin.
Figure 4 discloses the activity of CI inhibitor (C1IA) on carcinoma cells and non-malignant cells in culture using Leitz Cytophotometric measurement of cells on a Leitz MPVlCytophotometer mounted on an Orthoplan phase contrast microscope. By using Fluorescein Isothiocyanate (FITC) labeled specific polyclonal rabbit anti human CI inhibitor (anti C1IA) antibody (from Behring Institute, Marburg, Germany), using phase contrast microscope, one cell was centered into the measuring diaphragm of the Cytophotometer and the excitatation was measured for FITC activity. A number of 100 cells from each cell culture was counted.
The results were expressed in percentage of cells showing FITC conjugated anti CI IA binding, First column shows the distribution of percentage of CI IA "positive" primary carcinoma cell cultures, - showing specific binding of FITC conjugated rabbit anti human CI IA as evidenced by excitation measurements done from the first explantation of cells on micro-slides. The percentage-range of CI IA positive carcinoma cells in the cell cultures measured was plotted into a diagram and ranged from approximately 25% to >75%. Second column shows the distribution of carcinoma cell cultures sub-cultured in Falcon flasks and re-explanted on micro-slides in Leighton tubes and incubated in a humidified 37°C C02 incubator.
These measurements showed approximately the same distribution in percentage of CI IA positive cells. The third column show carcinoma cell cultures pre-incubated with unconjugated polyclonal rabbit anti CI IA, then washed times 3 in Phosphate buffered saline, (PBS buffer) at pH7.2-7.4 and re-incubated with FITC-conjugated polyclonal rabbit anti human CI IA. No cells showed any specific excitation. The fourth column incubated with FITC conjugated rabbit anti human CI IA and measured as described above showed no specific excitation when measured with the above described
Cytophotometer indicating no specific binding of anti CI IA on these non- malignant cells, which included among others human mesenchymal cells and fibroblast cultures.
Figure 5 discloses a 28 month old infant (patient No 1) who was treated with intravenous infusion of 180 ml (10% porcine semipurified IgG) of xenogeneic anti CI IA treatment ClIA prepared from pigs immunized with semipurified CI IA; CI IA and native C4 levels was monitored through the observation period where the infant was treated with chemotherapy and anti CI IA infusion.
Figure 6 discloses patient No. 1. Blood count of B lymphocyte changes in a 28 months old child with reticulosarcoma. Notice the significant increase of monocytes (MONO) immediately in connection with xenogeneic semipurified anti CI IA infusion, and Vincristine followed by a relatively high count of Monocytes followed by a sharp decrease of monocytes after the last cycle of Vincristine,xxxx On figure 4, it can be compared to the steady state of CI IA at the level of around 60+ mg/100 ml, but a constant increase of native C4. Immediately in connection with the infusion of the anti CI IA, a sharp increase of IgG lymphocytes are noted, which afterwards falls sharply during the Vincristine treatment. The increase in serum IgM after 9-14 days after the infusion of xenogeneiceic (porcine) semipurified Anti CI IA, indicating a possible active immunization against most probably porcine induced proteins.
Figure 7 discloses a 53 year old female with a disseminated mammary carcinoma with metastases to bone and to the resection zone on the skin, with
several cutaneous metastases en cuirass. As can be seen the patient started out with a CI IA in the blood of 80+ mg/100 ml and a native C4 at ~58 mg/100 ml. After the first xenogeneic (porcine) anti human semipurified IgG against semipurified CI IA the patient showed an initial minor increase in both CI IA and native C4, where CI inactivator within 1 month was around 55 mg/100 ml while her cutaneous metastases started to flatten. It was obvious that the Chemotherapy given around 8 days after over two cycles stopped the CI IA from decreasing further and native C4 started increasing. Then when the second and third xenogeneic sheep IgG against semipurified C1IA was infused the CI IA two peaks of blood CI IA was observed and at the same time after some irregular increases and dips in naaive C4 was observed ending with a sharp decrease in native C4 to around 2-10 mg/100 ml, and during the two months after, there was a steady decline in CI IA ending up at 30 mg/lOOml. After the second dosage of chemotherapy, native C4 showed steady increase ending up during the two month at around 70 mg/100 ml.
Figure 8 discloses the first (1.) increase of IgM as a most probable response on the first Xenogeneic (porcine) anti CI IA peaking after approx.. 2 weeks, and the second xenogeneic (sheep) anti CI IA peaking a few days to approximately 14 days after the second xenogeneic anti CI IA, showing what also is postulated in
this invention that patients cannot tolerate infusioin of xenogeneic proteins more than once, whereafter they start to produce IgM response. Figure 9a exemplifies an antigen -antibody reaction on the surface of a foreign cell or microorganism, followed by activation of the complement components, starting with binding Clqrs to the immune complex caused by the binding of the antibody to the antigen (e.g., of type IgGl, igG2, IgG3 or IgM). The Clqrs are split up into units and has activated and C4 whereof parts is bound to the surface of the "target" cell and split into other subunits, parts of C2 and parts of the complement components following the activation of C2 down to C9 is bound to the surface of the "target" cell. The multi components of C9 penetrates the plasma membrane of the cell or microorganism, and
extracellular fluid equilibrates with the internal of the cell, which then go into lysis.
Main effects of the complement reactions can be summarized into
"Opsonization" (enhancing "phagocytosis" of antigens, "Chemotaxis" - a mechanism attracting macrophages and neutrophils, "Cell Lysis" - rupturing membranes of foreign cells, and "Agglutination" - clustering and binding (sticking) of pathogens together
Over 30 proteins or protein fragments constitute the complement system, including serum proteins, membrane-like proteins, and cell membrane receptors. These proteins constitute 5% of the globulin fraction of blood serum and they are also serving as opsonins (an antibody or other substance that binds to foreign microorganisms or cells, making them more susceptible to phagocytosis).
Figure 9b discloses the CI Inactivator (CI IA) coat on the cancer cell membrane (the plasma cell membrane) is enlarged in this drawing in order for the reader to note the presence of the CI inactivator coat, and its inhibition and blocking of the activation of the Clqrs complex, which now cannot take place, whereby complement C4 will not be activated, which then results in no activation of C2 which means that the remaining classical complement activation ending with C9 will not take place. Therefore no Immune reaction, which includes the complement cascade, will take place on the Cancer cells coated with CI inactivator (C1IA). Antigen-Antibody cannot activate Complement CI for it to bind and activate complement component C4, (C4 is blocked). Therefore, no Complement Cascade will take place. The Result: no cancer cell lysis, no cancer cell death.
Figure 9c discloses the CI IA antigen coating the cancer cell is now exposed to the injected exogenous emonoclonal recombinant anti CI IA antibody (e.g., IgGi) in an optimal amount and titre will neutralize the CI IA coat on the cancer cells, so that the CI IA on the cancer cell cannot inhibit the activation of the complement component Clqrs complex, which again will bind and
activate complement component C4, which then will activate the remaining part of the complement system from C2 - C9 without interference from the now neutralized CI IA on the coat of the cancer cell. As can be understood, the antigen-antibody immune reaction Ag(Cl IA)-Ab(IgGl) is now able to activate the Complement Clqrs which now can activate C4 to C4b, and activate the C2 C9 Cascade. Result: Cancer Cell Lysis or Cell Death !
In this manner the treatment with recombinant anti human CI inactivator will then act as an external component infused into the patient and will among others, also target the cancer cells, coated with CI inactivator. Thus an exogenous mechanism is used to activate an endogenous, in vivo protein (CI inactivator), which is an induced antigen antibody effect to inactivate the CI inactivator and at the same time utilize the in vivo originated antigen reaction with the infused external antibody (e.g., external recombinant IgG) directed against CI inactivator, the binding of which will now bind Clqrs complex which will activate the C4 complement component. So by "luring" the coated CI inactivator working as a complement blockage, is now at the same time inactivated, and thereby hindered in any inhibitory influence of a Clqrs complex, whereby the C4 activation now really can be started by combining the exogenous anti CI inactivator antibody and the endogenous CI
inactivator, and the "lured" CI inactivator, bound to the exogenous antibody cannot block a complement reaction, which then in theory will take place ending up with binding of a "multicomponent" of C9 perforating the target cell, whereby the cell lysis will occur. Figure 9c is a theoretical schematic drawing showing how an injected exogenous humanized monoclonal recombinant antibody (e.g., IgGi) with its antigenic determinants will firmly bind to the CI IA coating the cancer cells. In this manner of activating the complement system by using an antibody to the CI IA coat on the cancer cells is in my opinion a unique and novel approach to indirectly hit the cancer cell plasma membrane with the complement cascade, leading to a most probable lysis and cancer cell death without utilizing another cancer related antigen, but by using the dual possibility of hitting the cancer cell with a complement cascade, is done in a manner, where the cancer cell
has lost its may be very important blocking protein, which otherwise is its way of escaping from being hit with the complement cascade system.
But now, where its defence mechanism through the CI IA coat is disarmed with the same weapon that has the dual function of primarily neutralize its escape mechanism against a complement activation attack, and at the same time the neutralizing antibody via its complement activating ability when bound to an antigen (CI IA) now can induce the complement activated lysis or death induced by the complement cascade towards the cancer cell.
Due to the fact that the protein itself, meaning the humanized recombinant anti CI IA monoclonal recombinant immunoglobulin which by binding to the antigen (CI IA) on the surface of the cancer cell now can activate the complement all the way from binding of the complement component CI qrs complex because of the lack of inhibitory effect from the neutralized CI IA coat on the cancer cell, because at the same time as this IgG such as for instance IgGi and other IgG molecules (minus IgG4) works as an antibody with determinants against CI IA. When applied to CI IA coated cancer cells it will neutralize the "blocking, masking" CI IA mechanism directly on the surface of the cancer cells and neutralize the CI IA coat on the cancer cells and immobilizing CI IA bound to the antigenic determinants from the humanized monoclonal recombinant anti CI IA antibody (IgGi), which now due to this antigen-antibody reaction will anti CI IA-C1 IA binding mechanics.
This will start the complement system to actively expose the cancer cell plasma membrane towards a possible lysis attack, exactly what this classical activated complement system will do, meaning that the cancer cell plasma membrane now will work as an alternate antigen, because this antigen- antibody binding of type IgG will activate the Clqrs complex.
Therefore, the binding of anti CI IA Igd and actually all other IgGs and also IgM (except IgG4), will then activate Clqrs complex, which again will activate C4; and C4 is already present in abundance due to the blocking of the circulating C4 by the increased CI IA, which now will lose its blocking or inhibiting effect of C4 and the rest of the complement system.
This will most probably elicit a cascade due to the neutralization of the CI IA coat on the cancer cells, neutralized by the injected or infused anti Human CI IA easily tolerated antibody leading to complement activation of the C1IA, now turning over to be an antigen, due to the infusion of an antibody specifically directed against CI IA and binding to the CI IA including binding to the surface of the cancer cells, inducing a resulting Clqrs activation because of the reaction between the infused anti CI IA. The CI IA especially coating the cancer cells is neutralized by the anti CI IA antibody (e.g., IgGl) and cannot hinder the activation of the Clqrs, which again activate the C4, which in cancer patients are found to be abundantly in place in the blood circulation due to the previous blockage of C4 activation because of the effect of the previous active, but now neutralized CI IA, especially on the cancer cells' coat or cell membrane also called the plasma membrane.
The CI qrs binding will now freely activate C4 which again will activate C2 and the classical complement reaction ending with C9 will take place and induce the lysis or death of the CI IA coated cancer cells. It is easy to monitor the dosage of anti CI IA infused at each treatment (which most probably have to be done several times of weeks to months).
Sequence listing
SEQ ID
SEQ ID NO: 1 Plasma protease CI inhibitor precursor [Homo sapiens] .
Signal peptide: aa. 1-22; mature peptide. Aa. 23-500 (NCBI Reference Sequence : NP_000053.2)
SEQ ID NO: 2 Plasma protease CI inhibitor precursor [Homo sapiens] .
Mature peptide
(NCBI Reference Sequence : NP_000053.2)
SEQ ID NO: 3 Signal peptide - homo sapiens serpin peptidase inhibitor, clade G (CI inhibitor).
Coding part
(NCBI Reference Sequence : NM_000062.2, coding part)
SEQ ID NO: 4 Homo sapiens serpin peptidase inhibitor, clade G (CI inhibitor).
Coding sequence, w/o signal sequence.
(NCBI Reference Sequence : NM_000062.2)
SEQ ID NO: 5 Hzec6
Vector
Items
1. A method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines.
2. The method according to claim 1, wherein said of recombinant CI esterase inhibitor is recombinant human CI esterase inhibitor.
3. The method according to any one of claims 1-2, wherein the HEK cell line is HEK 293T cell line (HumanCell, Naperville, Illinois, USA)
7. The method according to any one of the preceeding claims, wherein the human host cell is cultured to a cell density of above 103 cells per ml. 8. The method according to any of the preceeding claims, wherein
substantially no synthetic protease inhibitor(s) is/are added.
9. The method according to claim 7, wherein said CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
a. the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 b. a subsequence of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2;
c. a sequence which has at least 85% sequence identity with any one of the sequences set forth in i) and ii).
10. The method according to claim 7, wherein said sequences in ii) and iii) are immunologically equivalent to the sequence set forth in i).
11. The method according to claim 7 or 8, wherein said CI esterase inhibitor has a carbohydrate moiety, which constitutes from 45-50% of its molecular mass.
12. The method according to any one of claims 7-9, wherein said CI esterase inhibitor comprises sialic acid, such as in the same amounts as native CI esterase inhibitor. 13. The method according to any one of claims 7-10, wherein cells transfected with IgG to which the gene protope to CI IA antigen of the determinants of an IgG said sequences in ii) and iii) are capable of reacting specifically with an antiserum/polyclonal antibody or a monoclonal antibody raised against a polypeptide having the sequence set forth in i).
14. The method according to claim 11, wherein the monoclonal antibody is a mouse monoclonal antibody selected from the group consisting of: Clone 3F4- 1D9 (commercially available from Bio-Rad AbD Serotec, Lifespan Biosciences, Merck Millipore, Abnova and United States Biological) , clone LS-C39048 (commercially available form Lifespan Biosciences ) , clone [8D4C12E6 (commercially available from Sino Biological), clone 350507 (commercially available from R&D Systems), clone abxl0017 (commercially available from Abbexa), clone KT28 (commercially available from Thermo Fisher Scientific, Inc. and Abbiotec), clones 8F3 and 6C6 (commercially available from
Abbiotec), clone 10K343 (commercially available from United States
Biological), clone GWB-2F0410 (commercially available from Genway), clone 119-15582 (commercially available from Raybiotech), clone CAB-4608MH (commercially available from Creative Biomart), clone Bll (commercially available from Santa Cruz Biotechnology), clone MAA235Hu22 (commercially available from Cloud-Clone Corp), clones EPR8016, M81, EPR8015, ab54898 (commercially available from Abeam).
15. The method according to any one of claims 7-12, wherien the CI esterase inhibitor which comprises an amino acid sequence, which is a subsequence of the sequence set forth in SEQ ID NO. : 1, has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or the sequence set forth in SEQ ID NO. : 2.
16. The method according to any one of claims 7-13, wherien said said CI esterase inhibitor comprising a sequence, which has at least 85% identity to the sequences set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 or to a
subsequence thereof, has an inhibitory effect on the complement system and/or on plasma kallikrein, factor XIa, and/or factor Xlla, which is at least 50% of the inhibitory effect excerted by an equimolar amount of CI esterase inhibitor consisting of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
17. The method acocording to claim 13 or 14, wherein the inhibitory effect of said CI esterase inhibitors is measured in an in vitro assay comprising the steps of:
i) incubating from 2.5 ng to 1000 ng of the CI esterase inhibitors with 1 ng of factor Xlla in 20 μΙ of 50 mM Tris-HCI buffer at 37°C for 5 minutes;
ii) adding 1 pg purified prekallikrein (in 10 μΙ Tris-HCI) followed by incubation at 37°C for 1 minute;
ii) adding 20 μΙ Tris-HCI and 50 μΙ s-2302 chromogenic substrate for plasma kallikrein, FXIa and FXIIa (H-D-Pro-Phe-Arg-p-nitroaniline [pNA] -2HCI, DiaPharma Group, Inc., West Chester, Ohio), followed by incubation at 37°C for 10 minutes and addition of 50 μΙ acetic acid to stop reaction;
iii) determining kallikrein activity by reading the OD at 405 nm.
18. The method acocording to claim 13 or 14, wherein the inhibitory effect of said CI esterase inhibitors is measured in an in vitro assay comprising the steps of:
i) incubating 0.5 ml CI esterase inhibitor with 0.25 ml C reactive protein in 0.75 ml sodium phosphate buffer, ionic strength 0.15, pH 7.45 at room temperature for 15 minutes;
ii) adding 1 ml of 0.015 M N-acetyl-L-arginine methyl ester
hydrochloride (AAME) so as to produce a mixture of enzyme, inhibitor and substrate;
iii) removing a first 1 ml aliquot of said mixture in ii) and mixing said first aliquot with 1 ml 37 % formaldehyde;
iv) incubating the remainder of said mixture in ii) for 60 minutes at 37DC;
v) Removing a second 1 ml aliquot of said remainder of said
mixture, and mixing said second aliquot with 1 ml 37 %
formaldehyde; and
vi) measuring the titrable acidity in said first and second aliquot by addition of 0.05 N sodium hydroxide to an end point of pH 7.8. 19. The method according to any one of the preceding claims, wherein said CI esterase inhibitor consists of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2.
20. The method according to any of the preceding claims, wherein said exogenous nucleic acid sequence comprises a sequence selected from the group consisting of
i) SEQ ID NO. : 3 or SEQ ID NO. : 4 (NCBI Reference Sequence:
NM_000062.2)
ii) a subsequence of the sequence defined in i);
iii) a sequence which has at least 85% nucleic acid identity with the sequence set forth in i) or ii).
21. The method according to any of the preceding claims, wherein said
exogenous nucleic acid sequence encoding said CI esterase inhibitor has been inserted into a plasmid vector consisting/substantially consisting of the sequence set forth in SEQ ID NO. : 5 (Hzec6). PRODUCTION OF AB 's
24. A method for producingrecombinant human IgG and subtypes of IgG using the vector called pST2 for cloning and pST2-HF3 after cloning, linearized by Bglll and PciL transfected into HEK cells using the antigenic determinant against CI IA developed from the anti CI IA fab.
HUMAN HOST CELL
25. A human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the human cells such as HEK cells, PerC6 cells, amnion and an immortalised cell from the amniotic fluid, said cell comprising an exogenous nucleotide sequence encoding a CI esterase inhibitor.
22. The human host cell according to claim 20, wherein the cell comprises an exogenous nucleic acid sequence as defined in claim 18 and/or a plasmid vector as defined in claim 19.
23. The human host cell according to claim 20 or 21, wherein said cell expresses a CI esterase inhibitor as defined in any one of claims 7-17. 24. The human host cell according to any one of claims 20-23, wherein said host cell is a HEK 293 cell.
A RECOMBINANT CI IA
25. A recombinant CI esterase inhibitor obtainable by the method according to any one of claims 1-19.
COMPOSITION
26. A composition comprising a recombinant CI esterase inhibitor,
characterised in that
i) said composition comprises substantially no synthetic protease inhibitors, and/or
ii) said composition is serum free.
27. The composition according to claim 25, wherein said recombinant CI esterase inhibitor is as defined in claim 24. USE
28. A recombinant CI esterase inhibitor according to claim 24, or a
composition according to claim 25 or 26, for use as a medicament.
29. Use of a recombinant CI esterase inhibitor according to claim 24, or a composition according to claim 25 or 26, for the preparation of a medicament for the treatment and/or prevention and/or reduction of ischemia.
30. Use of a recombinant CI esterase inhibitor according to claim 24, or a composition according to claim 21 or 22, for the preparation of a medicament for the treatment and/or prevention and/or reduction of Ischemic stroke
(reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation).
31. Use of a recombinant CI esterase inhibitor according to claim 24, or a composition according to claim 25 or 26, for the preparation of a medicament for the treatment and/or prevention and/or reduction of Inflammation in spinal cord injury.
32. Use of a recombinant CI esterase inhibitor according to claim 24, or a composition according to claim 25 or 26, for the preparation of a medicament for the treatment of burn wounds (promotion of re-epithelialization,
prevention/alleviation of granulation tissue development and scar formation).
33. Use of a recombinant CI esterase inhibitor according to claim 24, or a composition according to claim 25 or 26, for the preparation of a medicament for the treatment and/or prevention and/or reduction of Hereditary
angioedema/ Quincke's edema.
34. The use according to any one of claims 28-32, wherein said CI esterase inhibitor or composition is administered intracerebrally, intraspinally, intrathecally, or intravenously. 35. A method for preventing, treating and/or alleviating Ischemic stroke
(reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI esterase inhibitor according to any of claim 24, or a composition according to claim 25 or 26.
36. A method for preventing, treating and/or alleviating Inflammation in spinal cord injury comprising administering to a subject in need thereof a
recombinant CI esterase inhibitor according to any of claim 24, or a
composition according to claim 25 or 26.
37. A method for treating burn wounds, Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI esterase inhibitor according to any of claim 20, or a composition according to claim 25 or 26.
38. The method according to any one of claims 34-3632, wherein said CI esterase inhibitor or composition is administered intracerebrally, intraspinally, intrathecally, or intravenously
AN ANTIBODY
39. An antibody raised against a CI esterase inhibitor according to claim 24.
40. The antibody according to claim 38; said antibody being a monoclonal antibody.
41. The antibody according to claim 38 or 39; said antibody being a polyclonal antibody/antiserum.
42. The antibody according to any one of claims 38 or 40, wherein said antibody is a polyclonal antibody raised against a composition comprising semi-purified CI esterase inhibitor.
43. The antibody according to claim 41, wherein said composition comprises one or more components selected from the group consisting of orosomucoid, alpha2 HS glycoprotein and Zn alpha2 glycoprotein, C-reactive protein. 44. The antibody according to claim 38 or 39, wherein said antibody is conjugated to a detectable marker.
45. The antibody according to claim 43, wherein said marker is a fluorescent marker.
46. The antibody according to claim 43 or 44, wherein said marker is a fluorescin derivative.
47. The antibody according to any one of claims 38 or 39, wherein said antibody is conjugated to a chemotherapeutic drug.
48. The antibody according to claim 46, wherein said chemotherapeutic drug is selected form the group consisting of chemotherapy drugs, alkylating agents and platinium drugs.
48a. The antibody according to claim 48, wherein the alkylating agents is selected from the group consisting of nitrogen mustards, such as
mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide
(Cytoxan®), ifosfamide, and melphalan, nitrosoureas such as streptozocin, carmustine (BCNU), and lomustine, alkyl sulfonates such as busulfan, triazines such as dacarbazine (DTIC) and temozolomide (Temodar®), ethylenimines such as thiotepa and altretamine (hexamethylmelamine).
48b. The antibody according to claim 48, wherein the platinum drug is is selected from the group consisting of cisplatin, carboplatin and oxalaplatin.
49. An antibody according to any one of claims 38-47 for use as a
medicament.
50. An antibody raised against a CI esterase inhibitor, wherein the CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
a. the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 b. a subsequence of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2;
c. a sequence which has at least 85% sequence identity with any one of the sequences set forth in i) and ii), 51. The antibody according to any one of claims 39-49 for use in the
manufacture of a medicament for the treatment of a malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas, kidney carcinomas bladder carcinomas, malignant brain tumors (e.g. glioblastomas or astrocytomas), astrocytoma, glioblastomas grade III-IV, gliomas, glioblastoma multiforme, oligodendrogliomas, ependymoma, and medulloblastoma.
52. The antibody for use according to claim 49, wherein said antibody is as described in any one of claims 38-46.
53. A method for treating a malignant carcinoma including malignant brain tumors (e.g. glioblastomas or astrocytomas), said method comprising administering to a subject in need thereof an antibody as defined in claim 49. 54. The method according to claim 51, wherein said antibody is as defined in any one of claims 38-47.
55. The antibody according to claim 46 or 47, for use in the manufacture of a medicament for treatment of a malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
56. The antibody for use according to claim 53, wherein the cancer is a malignant brain tumor (e.g. glioblastoma or an astrocytoma). DIAGNOSIS
57. A method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
e) a first parameter represented by the concentration of CI esterase
inactivator (CI IA) in at least one excreta from the individual and f) a second parameter represented by the concentration of complement component native C4 in at least one excreta from the individual wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
58. A method according to claim 57, wherein the first parameter is the total concentration of CI IA.
60. A method according to any one of the preceding claims, wherein the second parameter is the total concentration of native C4. 61. A method according to any one of the preceding claims, wherein the second parameter is the combination of the concentration of total native C4
62. A method according to any one of the preceding claims, wherein the at least one excreta is selected from the group consisting of blood, serum, saliva, spinal fluid, cerebro spinal fluid, pleura fluid, ascites fluid and urine.
63. A method according to any one of the preceding claims, wherein
concentration of CI IA and/or the concentration of native C4 is obtained any time before operation.
64. A method according to any one of the preceding claims, wherein
concentration of CI IA and/or the concentration of native C4 is obtained any time after an operation such as 2 weeks post-operation, 1 month post- operation, 1.5 month post-operation, 2 months post-operation, 3 month post- operation, 4 months post-operation, 5 month post-operation, 6 months post- operation, 7 month post-operation, 8 months post operation.
65. A method according to claim 59 or claim 61, wherein the combination is performed by logistic regression analysis.
66. A method according to any of the preceding claims, wherein the first discrimination value is determined by determining the total concentration of CI IA in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
67. A method according to any of the preceding claims, wherein the second discrimination value is determined by determining the total concentration of C4 in at least one excreta in both a healthy control population and a population with known cancer, thereby determining the first discriminating value which identifies the cancer population with a predetermined specificity or a predetermined sensitivity.
68. A method according to any of the preceding claims, wherein the
determination of the concentration is performed by means of an immunoassay or an active assay.
69. A method according to any of the preceding claims, wherein the
immunoassay is an ELISA. 70. A method according to any of the preceding claims, wherein the active assay is zymography.
71. A method according to any on the preceding claims, wherein the cancer is selected from the group consisting of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon
carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
72. Use of a method according to claim 57-71, for detection of early stage cancer.
73. Use according to claim 72, for detection of malignant carcinoma, squamous carcinomas (oesophageal cancer, larynx cancer, bronchial
carcinomas rectal carcinomas, pancreas carcinoma); adenocarcinomas, such as colon carcinomas of various types and sub types, pancreas carcinomas, ductal pancreas carcinoma, acinar pancreas carcinoma all glandular epithelial structures in which malignant adenocarcinomas such as breast carcinomas, bronchial carcinomas, ranging from lung alveolar carcinomas, breast carcinomas, e.g., ductal, lobular, etc., hepatocellular carcinomas and kidney carcinomas bladder carcinomas.
74. Use of a method according to claim 57-71, for monitoring the response to cancer treatment.
75. Use of a method according to claim 57-71, for monitoring the recurrence of a cancer. 76. A kit for performing the method according to claim 57-71, wherein said kit comprises CI IA and native C4.
77. A method of diagnosing rejection of transplants, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
b) incubating excreta from a human with anti-C4 and determining the
native C4 level in said sample
c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4 level with a second reference- level e) determining wither said human is likely of having cancer if the level of Cl-IA is at or above the first reference-level and if the level of native C4 is below the second reference-level.
A method of diagnosing cancer, said method comprising the steps of a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample,
b) incubating a blood sample or exsudate or transudate in connection with a suspected rejection of (transplanted) tissue from a human with anti-C4d and determining the C4d level in said sample, c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4d level with a second reference-level , e) determining whether said human is likely of experience a tissue or bone marrow rejection if the level of Cl-IA is normal or lower than 15 mg/100 ml and if the level of C4d is higher than 50 mg/100 ml. Examples
Example 1
As seen in figure 4, blood test from three (3) categories of patients were tested using rabbit anti human CI inactivator (BehringWerke, Marburg
Germany, now Behring Institute) and rabbit antibody against native (full molecule and not subunits of complement component C4). First category of individuals were "healthy subjects" from a Copenhagen based blood bank. Second category was blood samples from patients suffering from benign (non cancerous or malignant diseases), and the third category was blood samples from patients suffering from cancer.
In this investigation it was found that healthy subjects had in their I.V. blood an upper CI inactivator range of of approximately 40 mg/100 ml - and a lower CI inactivator concentration at > 15 mg/lOOml blood, and they had an upper naive C4 range of approximately 50 mg/lOOml - and a lower naive C4 range of ~ 10 mg/100 ml.
In the group of patients from a Copenhagen area hospital suffering from any kind of non-malignant disorders, such as patients from the departments of medicine suffering from heart or lung disease, and from miscellaneous diseases ranging from hormonal diseases, such as thyroid adenoma, various kidney diseases, and from department of surgery from the ER with various small traumas, such as musculo-skeletal disorders. These patients had in their I.V. blood an upper concentration CI inactivator of around 40 mg/100 ml and
a lower range of approximately 20 mg/100 ml. Their I.V. blood contained an upper level of naive C4 at 55 mg/100 ml and a lower level of approximately 10 mg/100 ml. In the group of patients from the cancer department of two Copenhagen hospitals, constituted any carcinomas and carcinoma metastases from various organs ranging from lung cancer, colon cancer, kidney cancer, bladder cancer, and patients with recurring carcinoma metastases. Their upper I.V. blood CI Iinactivator range was >90 mg/100 ml, and their lower blood CI inactivator was approximately 40 mg/100 ml (corresponding to the upper limit in healthy individuals with patients with non malignant disease. The naive i.v. blood C4 upper range was > 120 mg/100 ml and a lower range at approximately 45 mg/100 ml (corresponding to upper range of naive C4 in individuals with no malignant disease at 50-55 mg/100 ml).
So. Apart from the few healthy individuals or patients with non-malignant disease, who showed results of CI inactivator and naive C4 that showed overlap at their highest range of I.V. blood concentrations , with the lowest concentration of CI inactivator and naive C4 in patients with malignant cancer disease, the CI inactivator - and the naive C4 level found in cancer patients, indicate that by measuring the I.V. blood level of these two proteins, the majority of cancer patients showed higher levels of both CI inactivator and naive C4, which again suggest that this test combination may be a future indicator of a possible cancer disease, or active metastatic malignant disease making this test both a cheap monitoring system for a possible presence of cancer in patients, and may be an important indicator of remaining cancer in patients operated for a malignant cancer for instance of type, carcinoma.
Additionally, the very inexpensive test might be proven valuable when treating cancer patients with cytostatics or with monoclonal antibodies.
Of course the testing would be important, if and when cancer patients are treated with anti CI inactivator antibody or drugs that can lower CI
inactivator, and at the same time show whether the patient with cancer may
be considered in the beginning of remission or in remission by measuring both the presence of i.v. blood CI inactivator and naive C4 during the treatment. All in all a possibly useful tool to follow up any treatment against cancer.
There are two patients described in later examples who were treated with anti CI inactivator (semipurified porcine anti human CI inactivator and cytostatics) and the results on some of their immune system is shown both in regards to levels of CI inactivator and naive C4 in their I.V. blood during the treatment, and changes in their immune cells, such as lymphocytes shown in figures 4 to 8). The test system used to measure CI inactivator and naive C4 was at that time it was measured done by using Laurell Immunoelectrophoresis (Laurell CB, Analyt. Biochem. 1966, 15 :45-52).
Example 2
Figure 2 shows a simple overview of the classical pathway of the complement system with emphasis on the CI inhibitor or CI inactivator (CI IA) controls and inhibit the activity at the level of Clqrs complex in the classical pathway and in the lectin pathway. The higher relative concentration of CI IA present, when the Clqrs is trying to be dissociated to Clq Clr and Cls, the
dissociation of the Clqrs complex is inhibited from splitting up into Clq, Clr and Cls, and thereby the complement component C4 will be decreasingly dissociated to the opsonin part of the C4 and the C4, which is bound to the target in the case of a microorganism or a cell, part of the C4 should be bound directly to the cell surface and a part, called C4b, and a relatively C4a, a weak chemo attractant, is released.
Thus, Figure 2 is a simplified drawing of the complement system, targeting the classical pathway of a complement reaction, which is caused by an antigen- antibody reaction, and during chain of reactions occurring through the pathway until C9, the individual complement components are split up after activation to be bound to the pathogen, e.g., the cell, to perform opsonisation, meaning enhancing phagocytosis of antigen, chemotaxis which is attracting macrophages and neutrophils, ending up after sequential binding of part of the
split products of C4 to C9, where C9 consists of several fractions that results in a binding of part of the C4, C2, C3, C5 up to C9, catalyzing the enzymatic degradation of covalently bound complement proteins actually ending up by creating so called "pores" in the plasma membrane multiple part of the enzymatic molecule C9, resulting in cell lysis or cell death, in many cases eventually ending up with agglutination and further phagocytosis.
Based upon the definition of the various complement activations described above, it is the opinion of the inventor that among the 3 categories of pathways, the "classical pathway" of complement involved in antigen-antibody reaction on cells, is the pathway that has been blocked or inhibited on certain carcinoma cells due to the coating with CI IA of many of these cells hindering a disassociation of the Clqrs complex that again block the binding of the C4 molecule to the cell surface as well as the development of C4b, C4c and possibly C4d subunits. In this manner, the "classical pathway" of the
complement cascade ending up with C9, cannot take place, and thus protects the cancer cells from being lysed by this system. This should be compared to the observation at least on one patient suffering from metastatic breast cancer with cancer en cuirasse, which is defined as cutaneous metastasis, a
phenomenon that results from a tumor spreading via lymphatic or vascular embolization, direct implant during surgery or skin involvement by contiguity. The primary malignant tumor that most commonly metastasizes to the skin in women is breast cancer, which can be manifested through papulonodular lesions, erysipeloid or sclerodermiform infiltration, en cuirasse.
The findings of the CI IA indicating this powerful manner in which carcinomas can evade the immune defence system by avoiding the attack from the classical pathway of complement activation was evidenced by the first attempts to identify CI IA on human carcinoma cell cultures using the methods described previously by Osther et al. (Osther K and Linnemann R, Acta Pathol. Microbiol. Scand., 1973, Sect. B 81 : 271-272; Osther K and Linnemann R, Acta Pathol. Micrbiol. Scand. 1973, Sect B 81. 365-272) and one
on brain tumors (Osther K, Hojgaard K, Dybkjaer E, Acta Neurol. Scand. 1974, 50 :681-689).
Samples from malignant tissue were obtained during operation, transferred to sterile tubes containing Hanck's Stock solution, and kept at 4°C, brought down to the research laboratory, transportation time, did not exceed 3 hours. The cells were then cut from small biopsies, washed trypsinized and washed in Eagle MEM, centrifuged and explanted to obtain a monolayer on sterile micro- slides in Eagle MEM with fetal bovine serum in Leighton tubes for testing after 3 to 5 days post culturing, whereafter the cells on the microslides were tested for the presence of CI IA.
The cells from the same patients were also explanted in medium into 25 cc sterile Falcon flasks for monolayer culturing for longer periods. Control cells from benign tissue were obtained from patients with no overt cancer during surgery and processed as above. The cells cultured in Falcon flasks were later on also used for CI IA after some weeks, where a number of cells were removed by trypsination and explanted on new micro-slides for second or third testing for C1IA
The cells cultured on micro-slides in Leighton tubes were incubated with polyclonal rabbit anti CI IA - fluorescein isothiocyanate (FITC) conjugated (rabbit anti CI IA-FITC was obtained from Behring Werke (now Behring
Institute), Marburg, Germany. Non-conjugated rabbit anti CI IA were used for control experiments.
The measurement of 100 cells (one cell measured at a time) was done on a Leitz MPV I Cytophotometer system assembled on a Leitz Orthoplan
microscope. Excitation light source was an HBO 200 lamp house.
The Excitation light passed through a 1mm BG12 filter and a narrow band filter KP490 and a K530 barrier filter excluding non-specific signals. Borderline measurements between specific and non-specific fluorescence is described by Osther and Dybkjaer (Osther K, Dybkjaer E, Complement components on the surface of normal lymphocytes. Scand. J. Haematol., 1974, 13 : 24-32).
Control experiments using incubation of the cells with non-conjugated rabbit anti human CI IA, followed by consecutive washings in PBS buffer at pH 7.2- 7.4, followed by second incubation with FITC conjugated rabbit anti Human CI IA. The fluorescence measurement was plotted onto diagrams.
Figure 4 shows the primary carcinoma cell cultures measured after being incubated for 30 minutes at 37 degrees C, rinsed and measured in the Leitz microscop+e. The results of the measurement of several carcinoma cultures were plotted in and the percentage of cells showing FITC activity was plotted in to a table as shown in figure 4, several of the primary cell cultures sub- cultured from each primary carcinoma culture was explanted on micro-slides, incubated with FITC polyclonal anti CI esterase inhibitor (Behring Institute) and measured as described above.
The percentage of cells showing FITC activity were plotted in as a percentage of 100 cells. As the control of the specific binding carcinoma cells tested with rabbit polyclonal anti human CI IA FITC antibody (from Behring Institute, a duplicate of the carcinoma cells examined was tested for specific binding of the anti CI IA by running a matched duplicate of each carcinoma cell culture tested, which was pre-treated with unconjugated rabbit polyclonal anti CI esterase inhibitor also called CI IA (from Behring Institute), rinsed, washed times 3 with PBS at pH 7.2-7.4 and re-incubated with FITC conjugated polyclonal rabbit anti human CI esterase inhibitor also called anti CI IA, and the percentage of cells out of 100 tested was plotted in to the diagram.
The activity of CI inhibitor (ClIA) on carcinoma cells and non-malignant cells in culture was measured using Leitz MPV 1 Cytophotometer that could measure cells and show that the cells bound polyclonal rabbit anti CI IA - FITC (from Behring Institute, Marburg, Germany). A number of 100 cells from each cell culture was counted and FITC excitation was measured. The results were expressed in percentage of cells showing FITC conjugated anti CI IA binding, First column I figure 4 shows the percentage distribution of CI IA "positive" primary carcinoma cell cultures, - as measured on micro-slides,
which had been incubated with the antibody in a Leighton tube in a humidified 37°C C02 incubator. The percentage CI IA positive carcinoma cells in the cell cultures were plotted into the diagram. It appeared that around 25% to >75% of the carcinoma cells were found positive. Second column of the figure shows the distribution of carcinoma cell cultures sub-cultured in Falcon flasks and re-explanted on micro-slides in Leighton tubes. These measurements showed approximately the same distribution in percentage of CI IA positive cells. The third column show carcinoma cell cultures pre-incubated with unconjugated polyclonal rabbit anti CI IA, then washed times 3 in PBS buffer and re-incubated with FITC-conjugated rabbit anti CI IA. No cells showed any specific excitation. The fourth column incubated with FITC conjugated rabbit anti human CI IA and measured as described above showing no specific excitation when measured with the above described Cytophotometer indicating no specific binding of anti CI IA on these non-malignant cells, which included among others human mesenchymal cells.
As seen on figure 4, the percentage of FITC conjugated carcinoma cells both as primary explants and later as sub-cultured explants, showed a certain percentage of cells binding FITC conjugated anti CI IA (also called FITC polyclonal anti CI esterase inhibitor). Both the FITC conjugated polyclonal rabbit anti human CI IA and the un-conjugated polyclonal rabbit anti human CI IA was from Behring Werke, now called Behring Institute, Marburg,
Germany. The cells used for this measurement were explanted on micro- slides, subjected to 30 minutes incubation at in a C02 incubator at 37° C with FITC conjugated poly anti CI IA (BehringWerke, now Behring Institute), or initially incubated with un-conjugated rabbit polyclonal anti human CI IA and thereafter with FITC conjugated anti CI IA and measured as described above.
The percentage of carcinoma cells binding FITC conjugated rabbit anti CI IA ranged from around 25% to around or >75 % of the cells of the total of 100 cells measured from each culture. The binding of the FITC conjugated anti CI IA demonstrated according to the inventor a specific binding of anti CI IA to the plasma cell membrane of these malignant cell types, based upon the pre-
saturation assay, where the carcinoma cells were pre-incubated with unconjugated rabbit anti human CI IA, followed by 3 times of washing in PBS at pH 7.2-7.4, and re-incubated with FITC conjugated rabbit anti human CI IA from the same manufacturer (BehringWerke, Marburg, Germany). Non- malignant cell types consisting of mesenchymal human cell lines did not show any specific binding of FITC conjugated rabbit anti CI IA under the exact same condition as the carcinoma cell lines, (see Figure 4).
Patient No. 1
This infant was a 28 months old male with a malignant metastatic
reticulosarcoma. The events during the treatment has been referred to and interpreted the inventors present knowledge, which is somewhat different from previous descriptions and which exemplifies the theoretical effect of the neutralization of CI IA with these new methods that can be repeated, due to the fact that the present types of antibodies and test kits is an integrate part of this invention. As described previously like carcinomas, some sarcomas coincide with increasing CI inactivator and as it was also found in this patient the blood CI inactivator was at the first testing around 85 mg/100 ml; blood naive C4 was started immediately after surgery and at the time of infusion of porcine anti CI inactivator, at the normal level (compared to healthy individuals, etc.) of naive C4 concentration and a considerable decrease in CI inactivator coincided with the surgery, where a large tumor mass was removed and at the exact time where the patient was infused intravenously with porcine semipurified anti CI inactivator circulating blood CI inactivator increased immediately in connection with the infusion of porcine anti CI inactivator showed a sharp increase 2nd day following the anti CI inactivator and C4 also showed an simultaneous increase to over normal range
(compared to health individuals). Then the third day showed a decrease of blood CI inactivator but was still elevated compared to healthy donors, whereas C4 decreased to normal range. At the time when Vincristine was administered with some days' interval the CI inactivator concentration did show a steady fluctuating increase one month after anti CI inactivator was administered, and the C4 level steadily increased to higher than normal levels
and stayed elevated. One could postulate that it would have been an advantage, if the administration of Vincristine had started at least one (1) month after the infusion of anti CI inactivator. Due to the fact that when the infant was re-tested with cutaneous porcine immunoglobulin the patient appeared to show a positive reaction negating any further treatment.
Due to the fact that Vincristine has been described as having
immunosuppressive effect (see figue 6), this may be one of the reasons for the later persistent decrease in B lymphocytes (type IgG) later on through the repeat vincristine treatment. An interesting finding in the blood count was the increase of monocytes occurring after initiation and through the duration of the Vincristine treatment, such monocyte increase (>50) is normally observed in patients with lymphoma-like diseases and is related to a bad prognostic sign as described by (von Hohenstaufen KA, et al. Prognostic impact of monocyte count at presentation in mantle cell lymphoma. Br J
Haematol. 2013 Aug; 162(4) :465-73). The increase in IgM B lymphocyte after the porcine anti human Clinactivator coincides with the immune response in the patient due to the xenogeneic anti CI inactivator (see figures 5 and 6) : The changes of CI IA and eventually native C4 is described above (at the time of infusion with xenogeneic porcine semipurified anti human CI IA in the patient, with called a reticulosarcoma and also specified as a histiocytic lymphoma (this should be held together with the increase in the monocyte level and a decreased prognosis). This diagnosis was verified histologically approximately 7-8 month prior to the patient was treated with surgery and 8 months prior to the slow intravenous (IV) infusion of xenogeneic anti CI IA of 180 ml containing 10% semipurified IgG. Prior to this infusion the patient was treated with he was treated with chemotherapy combination consisting of Vincristine 3.5 mg administered during a 6 week period, combined with Cyclophosphamide, Streptonigrin given as the initial treatment at the first admission where he showed disseminated lymphomas, bone marrow
infiltration and marked thrombocytopenia. No hepatosplenomegaly.
During this period the lymphomas diminished, and the thrombocyte count normalized. One single bone marrow sample showed no malignant cells.
During the above described chemotherapy the patient developed rather severe infections. He also developed other side effects such as ataxia. Finally, when the patient developed severe cerebal edema no further cytostatic treatment could be given for a period of 2 months.
5-6 months after cessation of chemotherapy, the patient developed a fast growing cancer in his testicles. At this time the surgeon performed the removal of the large tumor mass as described previously, which coincided with a significant drop in blood CI inactivator
The first testing of blood CI IA was done at this time. A high level of CI IA, around 85 mg/100 ml was found (normal range 15 to 35 mg/100 ml).
Six weeks later his right testicle had grown so fast which was removed by a pediatric surgeon. Histology showed solid reticulo-sarcoma extending into the resection zone. The left testicle was somewhat enlarged but spared. At the day immediately after the surgery, the CI IA level decreased to below 70 mg/100 ml. The bone marrow revealed sarcomatous infiltration. A new drop in the thrombocyte count was treated with fresh platelet infusions. In order to avoid a possible disseminated intravascular coagulation, the patient was heparinized.
Sight (8) days after surgery the parents' consented in writing to an infusion with 180 ml 10% IgG xenogeneic (porcine) anti CI IA, produced in pigs immunized with semipurified CI IA administered slowly intravenously (IV), after that the cutaneous testing showed no reaction to the porcine antibody.
Blood testing for CI IA showed an initial immediate increase to around 90 mg/100 ml, which was theorized to be caused by lysis of cancer cells. Five (5) days after the infusion CI IA decreased to 50+ mg/100 ml and C4 decreased
from between 50 mg/100 ml (theorized normal range 20-40 mg/100 ml) to 20+ mg/100 ml.
Four (4) days following the porcine anti CI IA treatment was administered, the patient was re-started with Vincristine at a dose of 1.75 mg administered weekly. Already following the second dose of Vincristine, the left testis diminished. Bone marrow turned normal.
The Vincristin treatment had to be stopped at third dose because the patient re-developed neurotoxic symptoms, ataxia and paresis, but since this treatment, no serious infections were noticed.
Around two (2) months after ceased treatment with xenogeneic anti CI infusion followed by lower dose Vincristine the patient was monitored closely. The patient's CI IA did not show any tendency of decreasing during the period of Vincristine treatment and native C4 show a definite increase beyond 90+ mg/100 ml indicating a possible increased production of this C4 factor, It could be argued that the lack of decrease in CI IA and C4 was due to the immune-depression defect caused by the Vincristine treatment.
No lymphomas re-occurred, left testis was of normal size without sign of tumor. Bone marrow has been checked several times without any signs of tumor cells. Neither has scanning revealed any signs of relapse. Figure 6 shows fluctuations in immunoglobulin coated B lymphocytes and a definite significant increase in monocytoid (non-malignant) cells called
(MONO) appeared immediately after the anti CI IA infusion.
The high previously high level of IgG B lymphocytes declines significantly prior to anti CI IA infusion. An increase in IgG B lymphocytes increased
immediately after the infusion of anti CI IA and decreased significantly when second Vincristin dosage was given indicating the immunosuppressive effect of Viincristin. The following days (aroundlO - 14 days after the infusion with xenogeneic anti CI IA the IgM coated B lymphocyte count increases as a sign
of active i mmunization due to the infusion of the xenogeneic im munoglobulins.
The increase of the IgM B lymphocyte count after 10 - 14 days fol lowing the xenogeneic (porcine) im munoglobu lin indicate that it wou ld be contra- indicated to proceed with a second xenogeneic infusion with porcine proteins, which otherwise could have led to immunologica l or al lergic reactions in the patient. This is a clear indication of that even though a marked i mprovement of the tumor load which actually a lso resulted in no signs of tumor infiltrates in the bone marrow after the treatment.
This case presentation gives evidence of that one could not develop the anti CI IA treatment with xenogeneic anti CI IA, which meant that not until now where such complex human immunoglobulin against CI IA can be produced as a very important part of this invention providing a new development in using hu man or humanized anti CI IA for the im mune/complement inducing treatment of cancer patients in the future.
Example 3 Patient No 2
The second patient presented herein and whose blood CI inactivator and blood C4 is shown followed for 1 Vi month as seen in figure 7, a nd circulating B- lymphocyte followed in the sa me period seen in figure 8. The results has been re-evaluated by me with my present knowledge which is different from the previous description, and which exemplifies the necessity to arrive at novel ways of creating antibody against CI IA which can be used repeatedly in cancer patients aiming at a much more profound theoretica l effect. The use of being capable of producing hu man or hu ma nized anti CI IA antibody gives a whole new meaning to obtaining antigen-antibody reaction including a concomitant activation of the classical pathway of the hu man complement system, simply by neutralizing the blocking C1IA on the cancer cel ls and utilizing this antigen-antibody reaction as the activating mechanism of the classical pathway without being blocked by the neutralized CI IA. Therefore, in the neutralization of CI IA made in human antibody or humanized with
these new methods that can be repeated again and again and be monitored in a very simple manner by testing for the levels of CI IA and native C4 as base level and monitoring during the treatment, due to the fact that the present types of antibodies and test kits is an integrate part of this invention.
This 53 years old female suffered from disseminated metastases from mammary carcinoma. The patient was operated for around 1 - 2 years previously. Histiologically, roentgenological^ and scintigraphically verified osseous osteolytic metastases, bone marrow carcinosis and cutaneous metastases, deriving from a solid carcinoma.
The patient had previously been treated with irradiation against bilateral supra, infra and axillary lymph nodes, and palliative irradiation against lumbar column.
After the present relapse, the patient was initially infused slowly with xenogeneic (porcine) semipurified IgG anti human CI IA purified from pigs immunized with semipurified CI IA. The patient was infused slowly with an initial total dose of 680 ml (a 10% porcine IgG concentration) after testing negative to cutaneous test for porcine immunoglobulin. No significant temperature rise occurred.
One week after the initial treatment, the patient received two cycles (one week interval) cytostatic treatment with Vincristine (total dose 1.15 mg), 5- fluorouracil (total dose 600 mg), metothrexate (total dose 45 mg),
cyclophosphamide (total dose 150 mg) and prednisone (total dose 550 mg).
Twenty seven (27) days following the first xenogeneic infusion with
semipurified anti CI IA , a second and a third xenogeneic dosage was given each time with approximately 10% xenogeneic sheep antibody, because the patient showed positive skin test to porcine proteins, and negative for sheep protein. The semipurified IgG anti CI IA obtained from sheep of type Oxford after the animal had been immunized and boosted with semipurified CI IA.
This xenogeneic antibody was administered over two days with an interval of 7 days (total dose of the two cycles 580 ml with a immunoglobulin concentration of 10%). Maximum temperature rise 40.4. degree. C, which reverted after some hours to normal temperature level.
One month after last xenogeneic semipurified (sheep) anti CI IA infusion, the patient received a total of 5.0 mg Vincristine for a period of two months, 5- fluorouracil (1355 mg), and metothrexate (100 mg) and now a peroral administered dose of 70 mg cyclophosphamide is given daily.
Fifteen days after the initial xenogeneic (porcine) semipurified anti CI inactivator treatment (8 days after first cytostatic cycle) cutaneous
metastases flattened, and changed to horny scars. The osteolytic metastases were after a period of 3.5 months replaced by osteosclerotic structures. One bone marrow sample showed normoplastic marrow, another showed small islands of tumor cells surrounded by lymphocytes and connective tissue, and normoplastic marrow.
The patient is after a period of 3.5 months physically in good health. Alkaline phosphatases are normalized, BSR = 10 mm/hour, and the laboratory check shows normal condition.
The patient started out with a serum level of CI IA of approximately 82 mg/100 ml (normal range 15 to 35 mg/100 ml) and a serum native C4 of 58 mg/100 ml (theoretical normal range 20-40 mg/100 ml). Within in 24 hours after first infusion of xenogeneic (porcine) semipurified CI IA the level of serum CI IA increased to around ~95 mg/100 ml and native C4 increased to ~65 mg/lOOml. On day 8 after the infusion, and serum CI IA decreased to around 82 mg/lOOml and serum native C4 decreased to around 40 mg/lOOml prior the start of the combined chemotherapy incl. the Vincristine, serum native C4 decreased to around 40 mg/100 ml. After the first two cycles of chemotherapy
incl. Vincristine serum CI IA decreased to 55 mg/100 ml and was at the same level, when starting the second series of xenogeneic semipurified administered over two days anti CI inactivator, the CI IA immediately increased with two distinct repetitive increases followed by a gradually decline of CI IA,
concomitantly with a swift consumption of C4 as evidenced by an extremely low serum native C4 level around 10 mg/100 ml or lower, followed by an increase of serum C4 to around 40 mg/100 ml.
Around 3 weeks after the last anti CI IA infusion, a combined chemotherapy protocol with Vincristine,, 5 Fluorouracil and Methotrexate. This
immunosuppressive chemotherapy led to an increase of serum native C4 to 70 mg/100 ml, while the serum CI IA eventually during the same time span ended around 32-35 mg/100 ml as late as 3 months . After the cytostatic treatment C4 increased and CI inactivator stabilized.
During the second and third INA treatments, a fast significant rise of CI inactivator was noted simultaneously to a significant fall in C4. The
concentration of CI inactivator then began falling, and 3 months after first anti CI IA infusion, serum was CI inactivator was around normal standard values. C4 showed a rise to a normal titer.
One week after the first xenogeneic anti CI IA treatment, a relative B lymphocytosis of IgG type was noted (figure 8). At the second cytostatic cycle, the B lymphocytes dropped to subnormal values. At the time of second and third INA treatments, the patient revealed a slight B lymphocytosis of IgM, and simultaneously the IgG coated lymphocytes re-appeared at a normal range (IgM normal range 5-25%, IgG coated cells normal range 5- 20%). Three and a half months after first INA treatment, the patient has normal range of IgG and IgM coated lymphocytes.
As seen in figure 8, the thrombocytes dropped during the combined cytostatic treatment and INA treatment concomitantly to the development of purpura, treated with transfusions. Furthermore, a developed sinus tachycardia was
observed and treated with excellent effect.
Example 4
Measurement of CI IA on plated cells using Cyto photo meter.
As shown in figure 4 carcinoma cell cultures tested for the presence of CI inactivator as described previously represents a certain number of the cells using rabbit anti human CI inactivator-fluorescein isothiocyanate (FITC) labelled and tested using immunofluorescence microscope as described. The immunoflurorescence was measured arbitrarily by a Leitz MPV 1
Cytophotometer built on a Leitz Orthoplan phase contrast microscope with measuring window, which could measure one cell at a time, the result of which was written down. A number of 100 cells were brought into the diaphragm, one at a time, and the excitation value was written down. Controls was done by measuring cells incubated in Leighton tubes, starting with initial incubation of the cells in unlabeled rabbit anti human CI IA for 30 minutes at 37°C C02 incubator, washed times three in PBS buffer and then incubated with anti CI inactivator-FITC, the cells on the microslides were then washed x 3 with PBS, and measured using the Leitz MPV1 Cytophotometer microscope.
Benign cells did not show any significant binding as evidenced by no
measurable excitation after incubation with anti CI IA-FITC when measured in the MPV1.
It was evident for me that the cancer cells were producing the protein themselves, because when I trypsinized and washed the cells in PBS buffer at pH 7.2-7.4 from the corresponding or matched cancer cells subjected to the previous measurement and findings of CI IA by the anti CI IA-FITC was re- implanted on a new microslide in another Leighton tube, measured the same day using the same cytophotometer equipment, the trypsinized cells did not show any binding of anti CI IA-FITC, indicating that the trypsin had "cleaned" the plasma membrane of the cancer cell.
The cancer cells on the new microslide was cultured for approximately 3 days, and incubated with anti CI IA-FITC, a various percentage of the cancer cells were again binding anti CI IA-FITC indicating that the cells had reproduced the coating of CI IA on their plasma membrane. This measurement was performed in several carcinoma cell cultures from various types of carcinomas from various organs, from pleura fluid or ascites fluid drawn from patients suffering from metastatic carcinomas of breast, or from abdominal organs. The advantage using the humanized monoclonal recombinant IgG antibody (e.g., IgGi) with an optimal antigen determinant specificity binding firmly to the CI IA coated on the cancer cells will activate the complement component Clqrs complex, which again will bind and activate complement C4.
Claims
1. A method for the production of recombinant CI esterase inhibitor (CI IA), said method comprising culturing in vitro, under serum-free conditions, a human host cell transfected with an exogenous nucleic acid sequence encoding said CI esterase inhibitor, wherein the human host cell is selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the amniotic membrane, an immortalised cell from the amniotic fluid, HEK cell lines, CAP cell lines and CAP-T cell lines.
2. The method according to claim 1, wherein said of recombinant CI esterase inhibitor is recombinant human CI esterase inhibitor.
3. The method according to claim 1 or claim 2, wherein said CI esterase inhibitor comprises or consists of an amino acid sequence selected from the group consisting of:
a. the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2 b. a subsequence of the sequence set forth in SEQ ID NO. : 1 or SEQ ID NO. : 2;
c. a sequence which has at least 85% sequence identity with any one of the sequences set forth in i) and ii).
4. The method according to any of the preceding claims, wherein said exogenous nucleic acid sequence comprises a sequence selected from the group consisting of
i) SEQ ID NO. : 3 or SEQ ID NO. : 4 (NCBI Reference Sequence:
NM_000062.2)
ii) a subsequence of the sequence defined in i);
iii) a sequence which has at least 85% nucleic acid identity with the sequence set forth in i) or ii).
5. A human host cell selected from the group consisting of an immortalized cell from the placenta, an immortalized cell from the human cells such as HEK cells, PerC6 cells, amnion and an immortalised cell from the amniotic fluid,
said cell comprising an exogenous nucleotide sequence encoding a CI esterase inhibitor.
6. A recombinant CI esterase inhibitor obtainable by the method according to any one of claims 1-3.
7. A composition comprising a recombinant CI esterase inhibitor,
characterised in that
i) said composition comprises substantially no synthetic protease inhibitors, and/or
ii) said composition is serum free.
8. A recombinant CI esterase inhibitor according to claim 6, or a composition according to claim 7, for use as a medicament.
9. Use of a recombinant CI esterase inhibitor according to claim 6, or a composition according to claim 7, for the preparation of a medicament for the treatment and/or prevention and/or reduction of ischemia.
10. A method for preventing, treating and/or alleviating Ischemic stroke
(reduction of infarct volume, blood brain barrier damage, thrombus formation, edema formation, inflammation), comprising administering to a subject in need thereof a recombinant CI esterase inhibitor according to any of claim 6, or a composition according to claim 7.
11. A method for preventing, treating and/or alleviating Inflammation in spinal cord injury comprising administering to a subject in need thereof a
recombinant CI esterase inhibitor according to any of claim 6, or a
composition according to claim 7.
12. A method for treating burn wounds, Promotion of re-epithelialization, prevention/alleviation of granulation tissue development and scar formation comprising administering to a subject in need thereof a recombinant CI
esterase inhibitor according to any of claim 6, or a composition according to claim 7.
13. An antibody raised against a CI esterase inhibitor according to claim 6.
14. A method for treating a malignant carcinoma including malignant brain tumors (e.g. glioblastomas or astrocytomas), said method comprising administering to a subject in need thereof an antibody as defined in claim 13.
15. A method for detecting and/or screening and/or monitoring cancer in an individual, said method comprising determining :
a) a first parameter represented by the concentration of CI esterase
inactivator (CI IA) in at least one excreta from the individual and b) a second parameter represented by the concentration of complement component native C4 in at least one excreta from the individual wherein the presence of the first parameter at or above a predetermined first discrimination value and the presence of the second parameter below a second predetermined discrimination value is an indication that the individual has a high likelihood of having cancer.
16. Use of a method according to claim 15, for detection of early stage cancer.
17. A kit for performing the method according to claim 15, wherein said kit comprises CI IA and native C4.
18. A method of diagnosing rejection of transplants, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample
b) incubating excreta from a human with anti-C4 and determining the
native C4 level in said sample
c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4 level with a second reference-level
e) determining wither said human is likely of having cancer if the level of Cl-IA is at or above the first reference-level and if the level of native C4 is below the second reference-level.
19. A method of diagnosing cancer, said method comprising the steps of
a) incubating a sample from a human with anti-Cl IA and determining the CI IA level in said sample,
b) incubating a blood sample or exsudate or transudate in connection with a suspected rejection of (transplanted) tissue from a human with anti-C4d and determining the C4d level in said sample, c) comparing the determined CI IA level with a first reference-level, d) comparing the determined C4d level with a second reference-level, e) determining whether said human is likely of experience a tissue or bone marrow rejection if the level of Cl-IA is normal or lower than 15 mg/100 ml and if the level of C4d is higher than 50 mg/100 ml.
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