EP2293812A1

EP2293812A1 - Method for isolating cells and disease vectors from bodily fluids

Info

Publication number: EP2293812A1
Application number: EP09753532A
Authority: EP
Inventors: Hans-Werner Prof. Dr. Heinrich
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-05-30
Filing date: 2009-05-26
Publication date: 2011-03-16
Also published as: DE112009001907A5; DE102008025965A1; WO2009143816A4; US20110306034A1; WO2009143816A1

Abstract

The invention relates to a method, an arrangement, and the use thereof, on the basis whereof cells, including disease vectors, can be isolated from bodily fluids or analyzed using different separating methods. The aim is achieved by a method for isolating somatic cells, microorganisms, and/or viruses from bodily fluids of individuals, comprising the steps: (f) isolating immune complexes from the bodily fluid of an individual, (g) splitting the isolated immune complexes into the sub-units thereof, (h) bonding of the dissociated sub-units to a solid carrier, (i) incubating the modified carrier from (c) having cells or bodily fluid of the individual from (a), (j) isolating the incubated carrier from (d) having the cells, microorganisms, and/or viruses bonded to the sub-units and an arrangement particularly suited for patient-specific diagnosis and therapy, comprising – a carrier, preferably microparticles, - sub-units from immune complexes bonded to the carrier, preferably from CIC, of an individual, - cells, microorganisms, and/or viruses, preferably MNCs, from the same individual, bonded to the sub-units, and the use thereof.

Description

Process for isolating cells and pathogens from body fluids

The invention relates to the production of separation systems for cells and / or pathogens based on subunits from circulating immune complexes (CIC).

state of knowledge

The functional basis for the immune response is based on the finely tuned interaction of local and systemically effective cellular and humoral components of innate and acquired immunity. CIC are the product of actions taken and reactions between these components, e.g. Antibodies and antigens, receptors and associated ligands, complement factors, albumin and other plasma constituents. As a result, particulate agglomerates form, which are taken up and degraded by phagocytes. In immunopathological events, they are jointly responsible for the emergence and maintenance of a variety of acute and chronic inflammatory diseases.

CICs are the final morphological product and thus a mirror image of ongoing or expired processes of immune regulation. This applies to the physiological, life-sustaining immune response as well as immunopathological processes that can lead to disease and death. In every individual, CICs can be detected in the blood. Excessive values are e.g. associated with rheumatoid arthritis.

The analysis of the composition of the CIC by cleavage and determination of these subunits would be a snapshot of the immunoregulatory processes in an individual.

The subunits can be recovered gently. They retain their function of binding the respective reaction partner. The subunits can be up be coupled to a fixed matrix. This process is described in DE 19538641. It allows the removal of CIC and its subunits from the blood from the individuals from whom the CIC were obtained in the sense of affinity chromatography. In animal experiments with rats of the inbred strain BB / OK, an influence on immunoregulation by plasmapheresis could be demonstrated. BB / OK rats develop auto-aggressive ß-cell destruction similar to that of human juvenile diabetes (type 1 diabetes). CIC derived from plasmas were dissociated and covalently coupled to Sepharose. An extracorporeal and temporary treatment of rats in the prediabetic state with detectable insulitis (prerequisite for diagnostics in the human system) was able to arrest the process of β-cell destruction (Berg, S. et al., Diab. Stoffwechsel 11 (2002) Suppl 1. p. 55).

Pathogenetic basics, autoimmunity, allergies and tumors

Both the efficient defense of infectious agents and toxic substances and the homeostasis of healthy endogenous cells involve a number of immunocompetent cells and signaling systems. Antibodies, receptors and soluble mediators play an essential role in this complicated process. Antibodies are specifically directed against foreign and self-antigens and contribute to their elimination via various mechanisms (e.g., complement activation). The balance and proper function of the immune system depends on a number of factors.

Autoimmunity is characterized by the loss of tolerance to the body's own tissue. Multiple endogenous and exogenous factors are involved in the dysregulation of the immune system. Many mechanisms involved in the pathogenesis of autoimmune diseases are discussed. Cross-reacting antibodies or an antigen-driven specific immune response are two of the many possibilities. The inadequate control of the potentially autoreactive cells and the presentation of autoantigens eventually lead to the formation of autoreactive cells and the production of pathogenic antibodies, resulting in considerable damage and often life-threatening consequences. Autoantibody-mediated disorders form a broad spectrum ranging from the functional impairment of a receptor to destructive systemic disease.

The clinical picture depends on the specificity of the autoimmune reaction. Organ-specific diseases such as Graves' disease form one side of the spectrum. On the other hand, one finds the systemic autoimmune diseases, which are also associated with rheumatic diseases (eg rheumatoid arthritis). In this case, both lesions and antibodies are not limited to just one organ. Further, mixed and transitional forms are described, e.g. Myasthenia gravis.

As a consequence, the removal of the pathogenetically important autoantibodies or the CIC, which contain autoantibodies, is necessary to favorably influence the pathological processes and to control the associated symptoms.

Allergies also arise on the basis of a malfunction of the immune system.

Initial contact with certain antigens (allergens) induces false control in genetically predisposed persons in the balance between

Subclasses, IgE receptor distribution, TH1: TH2 ratio (and thus cytokine synthesis) and IgE synthesis. The result of this false control are the known acute or chronic symptoms after repeated allergen contact.

In addition to the increased IgE and IgE receptor synthesis, the result of the dysregulation is also a significant increase in IgG anti-IgE autoantibodies correlated with the serum IgE level, which can be detected in circulating immune complexes (thus, up to 32% of total serum IgE in patients with atopic dermatitis in the form of IgG anti-IgE immune complexes). However, there is no correlation between the concentration of IgG anti-IgE immune complexes and the severity of the disease, due to the known heterogeneity of the autoantibodies in their ability to induce histamine release from basophils.

Undoubtedly, autoantibodies play an immunomodulatory role that is not well known. In addition, IgG anti-IgE might be due to the high affinity of C1q IgGI and the Fc gamma receptor have an IgE-allergen complex eliminating function.

Previous approaches to a (more or less) causal therapy consist in the inactivation of free IgE, with the exception of the use of nedocromil, which probably prevents the subclass switch to IgE during B-cell maturation. In addition to the administration of high doses of donor IgG, which in some cases gives convincing results, the use of a recombinant humanized monoclonal antibody (rhuMAb-E25, a joint development of the companies Genentech, Novatis Pharma and Tanox Biosystems) has come the longest to be approved , This antibody binds to the Fc receptor binding region of the free IgE and prevents its docking with the different IgE receptors. This makes it possible to successfully reduce the concentration of free IgE by more than 95%. Elimination of the Mab-IgE complex occurs via the IgG-FcR binding pathway described above.

A drawback to the use of this antibody will be the cost problem, especially in patients whose IgE levels are greatly increased.

In addition to the inactivation of free IgE by antibodies, the extracorporeal removal could be a further, at least in certain patients promising method of therapy.

Attempts have been made in the former Soviet Union and Japan with immunoadsorbers that covalently bound specific anti-IgE antibodies as ligands. No side effects were observed. The concentration of free IgE was reduced by 83-98% and over a period of observation of 6 months, a therapeutic effect could be detected. Differentiated treatment protocols are unfortunately not available.

Tumors escape the immunological defense by releasing various factors that induce immune tolerance, as a result of which the indefinitely growing tumor is considered by the body's defense as an "embryo." Specific antibodies are formed against the tumor proteins called neoantigens. There are specific binding ligands. Antibodies such as ligands lead to the complexation of neoantigens. The immunological reaction is suppressed inter alia by the following mechanisms:

Release of immunosuppressive factors (such as soluble tumor necrosis factor).

5 receptor (sTNFRs) and neoantigens such as CEA, MUC1, CA15-3 and others). For some

Neoplasms, the blood concentration of neoantigen immunocomplex has prognostic significance.

• Influencing regulatory T cells (increase in TGFβ leads to the decrease of cytotoxic T cells)

I O • Increased ILT3 and ILT4 expression induced on dendritic cells

immune tolerance

• Antigen modulation

• Cytophilic antibodies mask tumor antigens

] 5 Loss of tissue integration resulting from increased expression of NFATtumorceiis (nuclear factor of activated T cells) and α-6-β-4 integrin; suppression of apoptosis (TOR - Sir3 - hsp; reaper - DIAP; Dbc2 protein - apoptosis; -) and / or increased cell division (HER-network - EGFR; ras - IL24 / IL24 receptor) and vascularization of solid tumors (CUGBP2 - COX2 -

20 prostaglandins) generate additional reproductive benefits for the growing tumor.

The condensation products of tumor action and body counteraction circulate as CIC's in the blood.

For more than 20 years, plasma exchange and protein A immunoadsorption have been used as a therapeutic measure in the treatment of tumors. A temporary, positive effect was detectable in certain cases.

viral infections

The interaction of virus and antibody usually leads to the neutralization of the virus. These virus immune complexes are taken up by phagocytes and reduced. However, some viruses, such as Dengue virus, are known to have virus infectious in immune complex even more infectious than free. Neutralized herpesvirus IgG immune complexes induce the same IL6 release in macrophages as free viral DNA. In dogs, a correlation was detected between the cerebellar immune complexes and the rheumatoid Artrithis (MAY et al., Rheumatology. 1994; 33: 27-31). Virus immune complexes play a pathogenic role in virus-induced immune complex diseases (eg, glomerulonephritis, Appel, GB, et al., NEJM, Feb. 18, 1993). Almost all patients suffering from chronic hepatitis C infection carry immune complex-bound HCV (MORITA T. et al., Hepato-gastroenterology, 1996, 43, 582-585), which can lead to permanent reinfection. A therapeutic influence of, usually chronic, viral infections with extracorporeal blood treatment procedures must therefore always remove the virus bound in the immune complex.

task

While the analysis of individual factors and their role in the physiological and pathological immune reaction generate a rapid gain in knowledge thanks to molecular biological examination methods, the investigation of regulatory relationships at the cellular level is still methodologically demanding.

The object of the invention is to provide a method, an arrangement and its use, on the basis of which, with different separation methods, cells, including pathogens, can be isolated or examined from body fluids. It is necessary in the first step to recognize the target cells.

Basis of the invention are CIC. They are not just the final product intended for degradation in the biological reaction chain. CICs are also key to isolating cells from the blood of individuals related to the current status of cellular immune regulation. Surprisingly, CIC subunits have been shown to bind to the surface of cells.

The task is solved according to claims 1 -20. Description of the invention

The CIC are prepared by known methods, e.g. Precipitation method or obtained by means of protein A - Adsorbem from the plasma of individuals. After such isolation, the CICs are broken down into their biologically active constituents. This is preferably done by lowering the pH to <3.0. Now the subunits can be prepared by known methods, e.g. Gel chromatography, separately and individually and mixed as a mixture of corresponding support materials, preferably microparticles, are suitable as solid support all available solid materials, in particular polystyrene or more preferably sepharose. With the aid of these "scavenger particles", cells can be separated from blood and other body fluids using different separation techniques In a second step, the cells can be characterized, cultured in vitro, subpopulated, manipulated, and used for further diagnostic or therapeutic purposes.

Cell depletion by means of a continuous or discontinuous extracorporeal method for therapeutic use can also be carried out using the method according to the invention. Such systems are also reactivated by known methods and possible for multiple use.

The invention is described in more detail with the following examples:

Example 1 :

Obtaining anticoagulated blood from donor JM using standard collection system. Centrifuge 4 ml of whole blood (10 min 600xg), remove 2 ml of plasma and add 2 ml of 0.1 M borate buffer. Add 4 ml of 7% PEG in borate buffer and vortex for 30 sec. Precipitation reaction overnight in the refrigerator.

Precipitation Centrifuge at 4 ° C for 30 min. 1600xg, remove supernatant and discard and wash the sediment with 10 ml of 3.5% PEG in borate buffer. Vortex 2 times for 1 min and centrifuge at 1600 C 4 ° C for 30 min. Remove supernatant and discard, take up sediment in 1 ml PBS pH 7.4 and homogenize solution.

Lower the pH of the CIC mixture to pH 3.0 with HCl.

5 protein coupling:

Activate polystyrene particles with NHS and EDC in MES buffer, wash activated particles with MES pH 3.0 and take up pH 3.0 in MES buffer, add protein solution, move solution, the proteins bind to the PS particles. Stopping of free binding sites by glycine or ethanolamines. Wash the PS particles with PBS and take up in PBS.

Incubation of the particles with cells:

a) Mono nuclear CeIIs (MNC) from donor JM to Ficollgradient

Take 3 MNC MNC in 300 μl PBS / BSA buffer pH 7.4 and place in a reaction tube containing 20,000 CIC 5 coupled particles. Incubate on a tilt roll mixer for 45 min at room temperature. Then make up the solution in the reaction tube with 3 ml of PBS-BSA buffer. Isolate the particles over the sieve and wash with 5 ml PBS / BSA buffer pH 7.4 and rinse with 1 ml PBS / BSA buffer pH 7.4 Particles in a multiwell plate 0 b) whole blood:

Centrifuge 2 ml of whole blood 350xg for 10 min and remove plasma and discard. Mix blood pellet with 1 ml PBS / BSA buffer pH 7.4 and centrifuge 350xg for 10 min. Repeat steps 2-3 4 times, remove supernatant and stir up concentrated whole blood. Add 1 ml of washed whole blood and 20,000 CIC-coupled particles to the 5 sample tubes and incubate on a tilt roll mixer for 45 min at RT. Fill the solution in the mixing container with 3 ml of PBS7BSA buffer and isolate the particles through a sieve and Wash with 10 ml PBS / BSA buffer pH 7.4. Rinse with 1 ml of PBS / BSA buffer pH 7.4 particles in a multiwell plate

Add Calcein AM and propidium iodide and evaluate on the fluorescence microscope.

Example 2:

Centrifuge 4 ml of whole blood (10 min 600xg), remove 2 ml of plasma and add 2 ml of 0.1 M borate buffer. Add 4 ml of 7% PEG in borate buffer and vortex for 30 sec. Precipitation reaction overnight in the refrigerator.

Precipitation 30 min 4 ⁰ C Centrifuge 1600xg, remove supernatant and discard and wash the sediment with 10 ml of 3.5% PEG in borate buffer. Vortex 2 times for 1 min and centrifuge at 1600 C 4 ° C for 30 min.

Remove supernatant and discard, take up sediment in 1 ml PBS pH 7.4 and homogenize solution.

Lower the pH of the CIC mixture to pH 3.0 with HCl.

Separation of a 3OkDa - 100kDa fraction:

Add to the protein solution in Amicon Ultra 100K with 4 ml PBS pH 2.7, centrifuge at 4000xg for 20 min at 4 ° C. Flow is transferred to an Amicon Ultra 3OK tube, centrifuged for 30 min at 4000xg 4 ⁰ C. Repeat steps 17 and 18 and wash protein solution in Amicon Ultra 3OK 2x with 4 ml of PBS pH 3.0. Combine 3OkDa-IOOkDa fractions and determine protein content. Protein coupling:

Incubation of the particles with cells:

a) Mono nuclear CeIIs (MNC) from donor HW to Ficoll gradient

Take 3 million MNC in 300 μl of PBS / BSA buffer pH 7.4 and place in a reaction tube containing 20,000 CIC-coupled particles. Incubate on a tilt roll mixer for 45 min at room temperature. Then make up the solution in the reaction tube with 3 ml of PBS-BSA buffer. Isolate the particles over the sieve and wash with 5 ml PBS / BSA buffer pH 7.4 and rinse with 1 ml PBS / BSA buffer pH 7.4 particles into a multiwell plate

b) Thoroughbred:

Centrifuge 2 ml of whole blood 350xg for 10 min and remove plasma and discard. Mix blood pellet with 1 ml PBS / BSA buffer pH 7.4 and centrifuge 350xg for 10 min. Repeat steps 2-3 4 times, remove supernatant and stir up concentrated whole blood. Add 1 ml of washed whole blood and 20,000 CIC-coupled particles to the sample tube and incubate on a tilt roll mixer at RT for 45 min. Fill the solution in the mixing container with 3 ml PBS7BSA buffer and isolate the particles through a sieve and wash with 10 ml PBS / BSA buffer pH 7.4. Rinse with 1 ml of PBS / BSA buffer pH 7.4 particles in a multiwell plate

Add Calcein AM and propidium iodide and evaluate on the fluorescence microscope. It can be seen in Figures 1-4 that with both the total CIC protein restriction and the 30-100 kDa fraction, MNC from the same donor can be isolated. At least some of these specific cells are not bound by antibodies, but by proteins with a relative molecular weight between 30 and 10OkDa.

Legend to the pictures

Figure 1 .: Total fraction of dissociated CIC coupled to particles and incubated with Mono Nuclear Cells (MNC) from donor JM to Ficoll gradient.

Figure 2 .: Total fraction of dissociated CIC coupled to particles and incubated with whole blood from donor JM.

Figure 3 .: 30-100kDa fraction of dissociated CIC coupled to particles and incubated with Mono Nuclear CeIIs (MNC) from donor JM to Ficoll gradient

Figure 4 .: 30-100kDa fraction of dissociated CIC coupled to particles and incubated with whole blood from donor JM.

All the features illustrated in the foregoing description, the appended claims and the drawings may be relevant to the realization of the invention in its various forms both individually and in any combination.

Claims

claims:

A method for isolating somatic cells, microorganisms and / or viruses from body fluids of individuals, comprising the steps

(a) Isolation of immune complexes from the body fluid of an individual

(b) splitting the isolated immune complexes into their subunits

(c) attachment of the dissociated subunits to a solid support

(d) incubation of the modified support from (c) with cells or body fluid of the individual from (a) (e) isolation of the incubated support from (d) with the cells, microorganisms and / or viruses attached to the subunits.

2. The method of claim 1, wherein in a further step

(f) characterizing the cells of (e), cultured in vitro, subdivided into subpopulations, manipulated, and / or for further diagnostic or therapeutic use

Purposes are used.

3. The method according to any one of claims 1-2, characterized in that the isolated by known methods, preferably by precipitation or from body fluids immune complexes are split into their subunits and bound in the dissociated state by known methods to solid support.

4. The method according to any one of claims 1-3, characterized in that the body fluid is blood, preferably anticoagulated blood or whole blood.

5. The method according to any one of claims 1-4, characterized in that the immune complexes are decomposed by a pH reduction, preferably to a pH <3.0, in their subunits.

6. The method according to any one of claims 1-5, characterized in that the subunits without further workup or after fractionation after Molecular weight or affinity adsorptively or covalently bound to solid supports.

7. The method according to any one of claims 1-6, characterized in that the solid support, preferably made of polystyrene, polyvinyl acrylate,

Polymethyl methacrylate, polymethyl acrylate, polylactides, or particularly preferably Sepharose formed, may have all forms, but preferably particles are used.

8. The method according to any one of claims 1-7, characterized in that all materials are suitable, which is predetermined by the purpose of a diagnostic or therapeutic application.

9. The method according to any one of claims 1-8, characterized in that conventional aids for the separation of the solid support and isolation of the solid

Carrier-bound cells, microorganisms and / or viruses are used, such as sedimentation, magnetic enrichment or separation by size difference and detachment of the cells by pH reduction or enzyme reactions.

10. The method according to any one of claims 1-9, wherein the steps (a) - (c) are characterized in that

(a) circulating immune complexes (CIC) can be obtained from the plasma of an individual by known methods, preferably precipitation methods or by means of protein A adsorbers

(b) after such isolation, the CICs are broken down into their biologically active constituents, in particular by lowering the pH to <3.0, and preferably the subunits are separated by known methods, in particular gel chromatography (c) the subunits individually or as Mixture to appropriate

Support materials, preferably microparticles, are coupled.

11. The method according to any one of claims 1-10, wherein the steps (a) - (c) are characterized in that

(a) anticoagulated blood is collected by means of a standard collection system, this whole blood is centrifuged, the plasma supernatant is removed, a plasma PEG precipitation reaction is performed, the precipitated

Centrifuged off and the CIC-containing sediment is taken up as a mixture in liquid

(b) lowering the pH of the CIC mixture of (a) to pH 3.0

(c) NHS and EDC activated polystyrene particles are incubated with the dissociated subunits of (b).

12. The method according to any one of claims 10-11, wherein the step (b) is further characterized in that after the dissociation of the CIC into its subunits, preferably by centrifugation a 3OkDa - 100kDa fraction is obtained therefrom, which then for the subsequent steps is used.

13. The method according to any one of claims 10-12, wherein the steps (d) - (e) are characterized in that

(d) the modified particles of (c) are incubated with Mono Nuclear Cells (MNC) of the subject of (a)

(e) isolating the particles from (d) with the attached MNC via a sieve.

14. The method according to any one of claims 10-12, wherein the steps (d) - (e) are characterized in that

(d) the modified particles of (c) are incubated with whole blood of the subject of (a), which has preferably been concentrated by centrifugation and removal of the plasma

(E) the particles from (d) with the attached cells, microorganisms and / or viruses are isolated via a sieve.

15. Arrangement, in particular suitable for use according to one of claims 16-20, which is preferably produced by a method according to one of claims 1-12, comprising

a carrier, preferably microparticles, subunits of immune complexes attached to the carrier, preferably from CIC, of an individual

cells, microorganisms and / or viruses, preferably MNCs, attached to the subunits from the same individual.

16. Use of the method according to any one of claims 1-14 or the

Arrangement according to claim 15 for the patient-specific diagnosis and therapy, preferably for the detection or treatment of pathological conditions of the individuals from whom the immune complexes have been obtained.

17. Use of the method according to any one of claims 1-14 or the

Arrangement according to claim 15 as an effective ingredient of products and processes suitable for use in an extracorporeal circuit.

18. Use of the method according to any one of claims 1-14 or the

Arrangement according to claim 15 for extracorporeal recovery or depletion of somatic cells, microorganisms and / or viruses recognized by immune complex components.

19. Use of the method according to any one of claims 1-14 or the

Arrangement according to claim 15 for the treatment of diseases which are triggered or maintained by dysregulation of the immune system, including certain chronic virus diseases.

20. Use of the method according to claim 19 in combination with the extracorporeal removal of immune complexes and / or their subunits and / or pro-inflammatory mediators.

Use of the method according to one of claims 1-14 or the arrangement according to claim 15 for the treatment of viral hepatitis, preferably hepatitis C.

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