Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

• the present invention relates to an in vitro method for the production of monoclonal antibodies, in particular of IgG type, making use of antigen presentation by dendritic cells.
• this invention describes a new approach for the generation of diagnostic and therapeutic antibodies against animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as for use against therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes.
• the approach is based on an in-vitro procedure that makes it possible to manufacture human monoclonal antibodies using the authentic antigen.
• a platform technology that enables the manufacture of monoclonal antibodies of human origin against a multitude of animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as for use against therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes and thereby dispenses with the classical, animal-intensive immunization model and hybhdoma technology scheme.
• pathogens whether they are bacteria, viruses, mycoplasma or other disease- causing toxins, agents or substances, have a direct effect on the survivability of the affected host.
• a direct immunization is not possible on account of the high toxicity of many pathogenic substances. Instead many toxoids have been employed whose toxicity is generally greatly reduced with aldehyde treatment. These detoxification reactions are effectively not controllable so that immunization occurs with surface epitopes on a relatively random basis.
• Similar animal-intensive tests aimed at the production of monoclonal as well as polyclonal antibodies against toxins have been reported several times. But despite the considerable effort, they were able to produce only a small quantity of antibodies or sera with an acceptable affinity to the native toxin.
• the antigen must then subsequently be internalized with CpG, the ligand for
• TLR 9 (toll like receptor 9) and then be processed and presented via class Il MHC (Ruprecht and Lanzavecchia, 2006).
• Th2 T cell stimulated with the same antigen must bind through its T cell receptor to the class Il MHC presented antigen on the B cell. This bonding is strengthened significantly by CD40/CD40L interaction.
• the technical problem underlying the present invention is to provide an improved in vitro method for the production of monoclonal antibodies, preferentially of IgG type.
• the present invention provides a method for the production of monoclonal antibodies, in particular of IgG type, against an antigen in vitro
• PBMC mononuclear cells
• step (c) cultuhng B cells and CD4+ T cells in the presence of the dendritic cells obtained in step (b) such that antigen presentation by the dendritic cells in the presence of the CD4+ T cells to the B cells leads to activation of the B cells to produce antibodies against the antigen;
• step (d) isolating one or more clones from the antibody-producing B cells obtained in step (c).
• the "B cells" used in step (c) will at least contain a population of na ⁇ ve B cells (potentially together with other populations of B cells such as B memory cells).
• the B cells employed in step (c) are at least about 60%, more preferred at least about 70 %, even more preferred at least about 80 %, most preferred at least about 90 % or more na ⁇ ve B cells.
• the CD4+ T cells used in step (c) at least contain a population of immature (or synonymously "na ⁇ ve") CD4+ T cells (potentially together with other populations of CD4+ T cells such as CD4+ T memory cells).
• the CD4+ T cells employed in step (c) are at least about 60%, more preferred at least about 70 %, even more preferred at least about 80 %, most preferred at least about 90 % or more immature CD4+ T cells.
• the above method further comprises the step (e) of immortalizing the one or more antibody-producing B cells clone(s) obtained in step (d).
• the immortalization may be carried out using Epstein-Barr-Virus (see Traggai et al., 2004), or may be accomplished by fusion of the clone(s) with myeloma cells (e.g. human myeloma cells, human immortalized primary myeloma cells, human-mouse hetero-myeloma cells, permanent human myeloma cells etc.).
• myeloma cells e.g. human myeloma cells, human immortalized primary myeloma cells, human-mouse hetero-myeloma cells, permanent human myeloma cells etc.
• the above step (b) comprises loading the dendritic cells with an
• step (b) is carried out in the presence of one or more Toll-like Receptor (TLR) agonists.
• TLR Toll-like Receptor
• Step (c) of the present invention may be carried out either by
• step (c1 ) cultuhng firstly the dendritic cells obtained in step (b) with CD4+ T cells in order to generate CD4+ T cells of type Th2 and then cultuhng secondly the obtained CD4+ T cells of type Th2 with B cells; or by
• step (c2) cultuhng the dendritic cells obtained in step (b) with CD4+ T cells and B cells simultaneously.
• step (c) of the inventive method is carried out as the above alternative (c1 ), which preferred method comprises the steps of:
• B preparing B cells from, preferably LLME-treated, PBMCs, more preferably at least enriching the sub-population of na ⁇ ve B cells (for example to values of percentages as outlined above), most preferably isolating na ⁇ ve B cells;
• CD4+ T cells from, preferably LLME-treated, PBMCs, more preferably at least enriching the sub-population of immature CD4+ T cells (for example to the values of percentages as outlined above), most preferably isolating immature CD4+ T cells;
• step (D) loading the (immature) DCs of step (A) with antigen (preferably by transfection as a complex of antigen and PEI-transferhn) under conditions so that the antigen is processed in the DCs and the processed antigen is presented on the DCs (mature DCs);
• antigen preferably by transfection as a complex of antigen and PEI-transferhn
• step (F) culturing the Th2 type CD4+ T cells in the presence of the B cells obtained in step (B) in order to activate the B cells to produce antibodies againt the antigen (this step is also called according to certain embodiments a "T/B (cell) dialog"); and (H) isolating one or more clones of the activated (antibody-producing) B cells.
• steps (A) to (C) can be carried out in any sequence or simultaneously.
• This sequential method especially provides for an effective maturation of DCs, presentation of antigen to CD4+ T cells and efficient activation of the B cells by the selected Th2 type CD4+ cells.
• a particularly preferred embodiment of this preferred mode of the method according to the invention is illustrated in Fig. 8.
• the analytical steps in Fig. 8 are optional as well as the time points indicated in Fig. 8 may be varied depending on the PBMCs (especially their quality) and the type and amount of antigen used as well as further factors known to the skilled person.
• the term "antigen" according to the present invention means any eptitope-containing object against which antigens can naturally be produced in an organism, in particular humans.
• the "antigen” may be selected from (human) pathogens, allergens, bacteria, toxins (such as endotoxin, enterotoxins etc.), mycoplasma, fungi and viruses as well as any parts of such antigens.
• Exemplary antigens are PrP (e.g. PrP peptide mix or recombinant bovine PrP.
• the B cells and/or the CD4+ T cells are isolated from, preferably LLME- treated, PBMCs, more preferably before employing them in step (c) of the present invention.
• dendritic cells in the development of an efficient in vitro immunization procedure according to the present invention for the generation of functional, human monoclonal antibodies is indispensable but has been neglected in investigations undertaken to date. In doing so the essential prerequisite for antibody production must logically be met: the relevant antigens must be captured by the endogenous processing of the dendritic cells and thereby ensure the attainment of an optimal and efficient class Il MHC antigen presentation. Should an "escape" of the natural antigen occur from the endosomal compartment into the cytosol, which would lead to the result of an undesired cross presentation of the antigen by way of class I MHC, the antigen must be "forced" into the exogenous processing modus by means of speficic surface modifications.
• Monocytes were identified as the principal stem cell precursors, which differentiated into immature DCs after the administration of GM-CSF and IL-4, which in turn are capable of efficient antigen intake (Peters et al., 1993). Further it was shown in recent years that the application of IL-15 instead of IL-4 effectuated the differentiation of the monocytes into immature dendritic cells that showed characteristics of
• the immortalization of human B cells occurs frequently with less efficiency and loss of antigen specificity on account of the lengthy cultivation process.
• a fusion occurs with humanized and human-immortalized primary myeloma cells, mixed human-mouse cells or via permanent human myeloma cells including those that have been modified through physical, biochemical or genetic engineered processes.
• the immortalization of the primary myeloma cells can be achieved in this connection using natural, biochemical, genetic engineering or physical processes. It is important that the fused cell lines can express and secrete typical and functional monoclonal antibodies.
• the cultivation of individual antibodies secreting cell clones occurs using specific, generally recognized cell cultivation procedures as well as optimized procedures used for commercial and industrial applications.
• the harvesting of antibodies occurs using the current state of the art, optimized laboratory techniques.
• the invention is based on a novel technology that is based on dendritic cells and with which in vitro monoclonal antibodies (also human) for use against any antigen including animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their
• the antigen e.g. a toxin
• the antigen can be used in its native form
• the pathogenic substance is a substance that causes the pathogenic substance:
• monclonal antibodies will be used that are either anti-toxoid sera with cross-reactivity to the toxin or are aimed against recombinant subunits of the relevant animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes.
• In vitro immunization is a procedure that was first described about 20 years ago (Borrebaeck, 1989) and that is based on the incubation of isolated human PMBCs with peptides that are derived from different proteins. As a rule this method delivers antigen specific IgM within two to three months, but an efficient Jsootope switch"to IgG has not been observed under this method (Xu et al., 2004). It is suggested to use this method for in vitro immunization as a positive control measure.
• donor PBMCs will be treated with LLME (L-Leucyl-L-Leucin Methylester) to remove monocytes, cytotoxic T cells, CD8+ suppresor T cells and NK cells which, as shown in earlier studies, markedly inhibit antibody production (Aiba et al., 2006).
• the LLME-treated PBMCs are then incubated with the corresponding peptide or antigen in the presence of different cytokines (IL-2, IL-4, IL-10). Then the in vitro immunized B cells are immortalized with Epstein-Barr-Virus (EBV) and cloned biologically in order to find high-producing B cell clones.
• EBV Epstein-Barr-Virus
• MDDC monocyte-dehved dendritic cells
• MDDCs with soluble monomers of animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by
• biochemical, chemical and physical processes monomers do not lead to an efficient class Il MHC presentation, care must be taken by specific ..retargeting" strategies with the aid of loaded polykations, which can be coupled with ligands for specific cellular receptors, to ensure that animal and human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by
• TLRs 7/8 and 9 appear to play an important role in this respect.
• TLRs bind in the endosomes to the released viral genomes.
• TLRs 7/8 appear to be responsible for binding to single-stranded RNA genomes, while TLR 8 binds to unmethylated CpG motifs in viral DNA genomes
• TLRs 7/8 ssRNA
• CpG-ODN CpG-ODN
• Th2 type CD4 + helper T cells is of prime significance for the induction of an adaptive humoral immune response.
• An essential requirement for this is the class Il MHC antigen presentation by dendritic cells as well as the regulation of the costimulating surface molecules CD40, CD80, CD83, CD86 and OX40, typical for matured dendritic cells.
• the cytokine milieu is of great importance.
• Fig. 1 shows a schematic representation of an in vitro method for the production of monoclonal antibodies according to the prior art (see Borrebaeck, 1989).
• Fig. 2 shows a schematic representation of an in vitro method for producing
• FIG. 3 shows a schematic representation of the induction of a B cell-dependent T cell response using the prion protein (PrP) as antigen.
• PrP prion protein
• T/B dialog is induced thus leading to the production of antigen-specific IgG.
• Fig. 4 shows graphical representations of the results of FACS analyses of DC
• A expression of HLA-DR and CD14 markers on PBMCs (day 0), immature DCs (day 4) and mature DCs (day 6).
• CD14 expression is strong on day 0, and weak on days 4 and 6.
• B expression of HLA-DR subpopulations on PBMCs (day 0), immature DCs (day 4) and mature DCs (day 6).
• C expression of
• CD80/CD86 on immature DCs (day 4) and mature DCs (day 6). CD80/CD86 negative DCs are observed on day 4, but not on day 6. After the addition of autologous T cells expression of CD 80 is increased.
• Fig. 5 shows a schematic representation illustrating the receptor-mediated
• Fig. 6 shows a graphical representation of IFN- ⁇ ELISA measurements of DC
• PEI-Tf PEI-transferrin
• ME mite extract
• Fig. 7 shows photographs of microscopic specimens of mature DCs/T cell co- cultures demonstracting their interaction (immunologic synapses are indicated by an arrow in each panel).
• Fig. 8 shows a preferred embodiment of the in vitro immunization protocol according to the invention.
• the starting cell population from which the dendritic cells and the cells that serve to generate the B cell clones are produced are peripheral blood mononuclear cells (PBMC). These are produced from “buffy coats” or from fresh blood of human donors by means of density gradient centhfugation using lymphoprep. After homologation of the expression status of the surface markers CD3, CD4, CD8, CD14, CD19, CD20, CD25, CD26 and CD34, the PBMCs are cryopreserved with the number of cells at 10 7 in the presence of of DMSO and as autologous serum cryoconserved and stored on liquid N 2 .
• PBMC peripheral blood mononuclear cells
• cryovials each containing 5 x 10 7 cells from 1 liter blood are obtained.
• Important for the quality of PBMC cultures is the selection of the appropriate cell density of well-suspended cells in the step of spreading the cells.
• a culture in 24-well tissue culture plates (CorningStar) using 1 to 1.5 ml of cell culture per well at a cell density of 1 x 10 6 cells has been determined as highly preferred
• Serum-free DC medium (Cellgenix), supplemented with IL-2, IL-4 and IL-6, was used as culture medium.
• the PBMC cultures were treated with Leu-Leu methylester hydrobromide (LLME) in order to remove natural killer (NK) cells and cytotoxic T cells.
• LLME Leu-Leu methylester hydrobromide
• NK natural killer cells
• cytotoxic T cells Corresponding cultures were stable for about 9 to 10 days.
• Thus-obtained PBMC cultures were stored in the presence of DMSO under liquid nitrogen which had no adverse effect on culture quality.
• Example 2 Purification and characterization of the lymphocyte
• CD4 + T cells Here particularly the CD4 + T cells are of special importance. Therefore the CD4 + T cells are isolated from the aggregate population of PMBCs using appropriate compartmentation kits made by the Miltenyi company (Untouched T cell kit). Using FACS analysis for the CD4 marker, a yield of 90% T cells was observed by using this kit. The cell population thereby purified is then examined as to its immunological status. For the CD4 + T cells the Th1/Th2 proportion is particularly important and whether "memory" CD4 + T cells are existent. This is to be examined by means of proliferation tests using a recognized method (Voss et al., 1992; 1993; Dittmer et al., 1994; Weber et al., 2001 ).
• TT tetanus toxoid
• PHA Phytahaemagglutinin
• JCV human Polyomavirus JCV
• the cytokine pattern obtained will provide information about the composition of the CD4 + T cell population.
• B cells The B cells are similarly isolated from the aggregate population of the PBMCs using appprophate compartmentation kits made by the Miltenyi company. As with the CD4 + T cells, the B cell population is examined as to the ratio of ,,naive" B cells to ..memory" B cells. This is done by the analysis of defined surface markers using flow cytometry. The expression pattern of the surface markers will give information on the ratio of "naive" B cells to "memory” B cells. Parallel to this the serological status of the donors will be ascertained.
• MDDCs Loading of MDDCs with animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes or the corresponding pathogen/tumor antigen polymer complex in varying concentrations.
• MDDCs were loaded with prion protein (PrP) as antigen, in particular bovine prion protein peptide mix (PrP mix) and recombinant bovine prion protein were used.
• PrP prion protein
• PrP mix bovine prion protein peptide mix
• ME Mite extract
• the DCs were FACS analysed for expression of the markers HLA-DR/CD14 (Fig. 4A, B). CD80/CD86 (Fig. 4C) and CD83/CD1 a (not shown).
• the expression of marker HLA-DR/CD14 was determined by FACS on monocytic progenitor cells.
• Expression of the markers CD80/CD86 (specific for DC cells) was analysed by FACS on immature DCs on day 4 of culture and on mature DCs on day 6 of culture (Fig. 4).
• a decrease in CD14 expression by simultaneous increase in HLA-DR expression could be observed (Fig. 4A and B) indicated a maturation of the DC cells.
• the immature DCs were loaded with antigen on day 4 together with supplementation by TNF- ⁇ , IL-1 ⁇ and IL-6. Upon this treatment, the proportion of CD80/CD86 positive DCs increased from day 4 to day 6.
• the maturation of DCs is dependent on the uptake of the antigen. Since antigens show different biochemical characteristics (such as, for example, net charge) the selection of the optimal transfection agent should preferably determined for each antigen. Measurement of the zeta potential using the Zetasizer (Malvern) showed a potential of -37.8 mV for ME and +26.4 for recombinant PrP. In the present specific embodiment the polycation PEI- transferrin (BenderMedSystems) was used as the transfection agent. The ratio of PEI-transferhn to agent was 1 :5.2. CpG DNA which can also act as a danger signal (see Fig. 5), was used together with PEI-transferrin in the case of recombinant bovine PrP. 4. Determination of IFN- ⁇ / ⁇ / ⁇ as well as IL-6, IL-10 and IL-12 secretion using different periods of time.
• immune complexes (IC) of animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes are used for loading of the MDDCs.
• IC immune complexes
• For this immune sera are used against the animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes.
• the loading of the MDDCs and further examination of maturation and trafficking are carried out as described above.
• VLP virus like particles
• the nucleic acids are packaged in virus like particles (VLP).
• VLP virus like particles
• the VLPs are loaded with the identical polymers as the coapplied animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes in order to assure uptake into the identical cell compartment.
• the dendritic cells are incubated with the naive CD4 + T cells in different ratios (e.g. 1 :50; 1 :100; 1 :200,1 :400; 1 :800).
• a 5 to 20fold surplus of T cells to (mature) DCs (prepared as outlined above in B.3.) is used.
• the specific ratio of T cells to DCs is in part dependent on the quality of the PBMCs.
• Th2 T cells The secretion of cytokines IL-4, IL-5, IL-6, IL-10, IL-12 and IFN- Y is observed over time. In addition the development of the Th2 surface marker CCR2, 3 and 4 is observed. The generation of Th2 T cells is carried out as well in the presence of antibodies against IL-12 and IFN- ⁇ (Th 1 -promoting cytokines). In addition, research is to be done as to whether the generation of Th2 cells can be improved in the presence of IL-4, IL-6 and IL-10. In a specific embodiment for determining whether the immune reaction has a Th1 or Th2 character, the concentrations of IL-10 (indicative for Th2) and IFN- ⁇ (indicative for Th1 ) are measured by ELISA.
• the T cell costimulatory markers CD80/CD86 showed a different expression pattern as compared to the cultures without addition of T cells (Fig. 4C): while CD80 increased strongly, CD86 expression decreased notably. It is believed that this expression pattern is either due to an increased IL-10 release resulting in a selective inhibition of CD86 expression, or due to the time dependency of CD80/CD86 expression (CD86 is expressed before CD80). Since differences in CD80/CD86 expression between the samples that received antigen (4 ⁇ g/ml ME) and the negative controls could not be observed, it is also contemplated to use the addition of CpG DNA as a danger signal in order to selectively increase the expression of CD86.
• naive B cells are isolated from PBMCs by negative selection (Miltenyi) in highly purified form.
• the B cells are incubated with the different forms of animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes, together with different TLR 7/9 agonists.
• the activation is promoted by costimulation with anti-CD40 antibodies, or specific Th2 helper T cells of animal and human pathogen, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes, in different ratios.
• anti-CD40 antibodies or specific Th2 helper T cells of animal and human pathogen, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes, in different ratios.
• cytokines such as IL-2, IL-4, IL-6, IL-10 and IL-21 are examined alone or in combination with respect to activation of the B cells.
• the cytidine deaminase (the key enzyme for the "isotype switch") is set using real time PCR.
• Fig. 8 shows a specific embodiment of the synthesis of the individual steps: at day 0 PBMCs stored under nitrogen are thawed. For DC maturation and treatment with LLME three 2 ml cryovials each containing approximately 5 x 10 7 cells are used for each process. Analytical methods (FACS, ELISA, ELISPOT and Western Blot) are indicated in Fig. 8. The time periods indicated in Fig. 8 refer to the quantitative cell measurements in the FACS device or using the ELISPOT analysis. Cytokines and immunoglobulins are measured in pre-centrifuged cell supernatants with ELISA tests. These measurements are preferred for monitoring the induction of a B cell-dependent T cell response, but are not essential for the function of the protocol per se. When the complete protocol is routinely established for a given antigen, such control measurements can be reduced or even be avoided making the principle protocol much more uncomplicated.
• the isolation, maturation and transfection of DCs has been optimized with respect to incubation times and individual time points as indicated in Fig. 8. Also the optimal time point for the addition of T cells (1 h after transfection of DCs with antigen) has been determined by measurement of IFN- ⁇ .
• the time point for the addition of immunized PBMCs is preferably not before day 5.
• the addition of immunized or non-immunized B cells may increase the number of antigen-positive B cells.
• ME induced a B cell-dependent T cell response.
• the activated B cells are isolated from the corresponding system and immortalized with a new method (Traggiai et al., 2004). To this end the isolated B cells are restimulated with the appropriate antigen and corresponding TLR agonists and immportalized with Epstein-Barr- Virus.
• the dissociation constants of the purified antibodies is determined in competition tests first. Then the diagnostic and neutralizing capacity of the purified antibodies is measured. Different biological tissues and liquids with different concentrations of animal and human pathogen, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins, including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes are ,,spiked" for the diagnostic examination. The antigen identification occurs by means of the purified antibodies in the ELISA.
• the chosen animal or human pathogens, bacteria, mycoplasma, virus particles, viruses and their exotoxins, endotoxins, and enterotoxins including their modifications occurring genetically and by biochemical, chemical and physical processes, as well as therapeutically relevant proteins and tumoral agents, including their modifications occurring genetically and by biochemical, chemical and physical processes are is incubated in the presence of known different antibody
• Zafiropoulos, Andersson, Krambovitis and Borrebaeck Induction of antigen-specific isotype switching by in vitro immunization of human na ⁇ ve B lymphocytes. J.
• Hunsmann, Weber and Luke Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: Formation of virus-like particles usefull for immunological and therapeutic studies. J. Virol. 73: 4465-4469 (1999) Luke, Petry, Ast, Wilke, Goldmann, Wagner and Schnabelrauch: Composition for transferring active compounds in a cell-specific manner. DE10131145A1 (filed 2001 ), EP1270586B1 (date of patent 2006), JP2003061693 (filed 2003), US2003044961A1 (filed 2002) Petry, Goldmann, Ast and Luke: The use of virus-like particles for gene transfer. Curr. Opin. MoI. Ther. 5: 524-528 (2003)
• Voss, Dittmer, Coulibaly, Makoschey, Petry, Luke and Hunsmann Differences in the B and T cell immune response to the envelope glycoprotein 130 (gp130) of the macaque strain of simian immunodeficiency virus (SIVmac), induced by immunization of rhesus macaques with virus-derived or vaccinia virus-expressed gp130.
• SIVmac simian immunodeficiency virus

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