SE461985B - IN VITRO IMMUNIZATION OF Lymphocyte-containing CELL POPULATIONS AND MATERIAL KIT - Google Patents

Abstract

A method for increasing the yield of antigen-positive hybridomae and the yield of IgG-producing hybridomae amongst antigen-positive hybridomae in in vitro immunisation of lymphocyte-containing cell populations, is described. In the method, lysosomotropic agents are caused to act in vitro on lymphocyte-containing cell populations for the removal of cell populations with a negative effect on the in vitro immunisation, whereupon the lymphocytes are in vitro immunised with thymus-dependent or thymus-independent antigens in the presence of a polyclonal activator. If a well-adjusted quantity of a polyclonal activator is used, it is possible to obtain an increase only in the yield of IgG-producing hybridomae amongst antigen-positive hybridomae in in vitro immunisation without a lysosomotropic agent. A kit for use in in vitro immunisation of lymphocyte-containing cell populations is also described. Said kit comprises at least three containers, of which one holds lymphokines as active constituent, the second holds lysosomotropic agents as active constituent, and the third holds a polyclonal activator as active constituent. Another kit according to the invention does not have the container with lysosomotropic agents.

Description

461 985 10 15 20 25 30 35 2 years and there are now well-developed in vitro immunization systems.

However, what characterizes these systems is that they predominantly result in a primary immunization with the production of IgM-type antibodies. Various methods have been tested to bring about a transition from IgM to IgG production in an in vitro system, which occurs in vivo naturally for most antigens.

For most antigens, current in vitro immunization techniques provide a good yield of specific hybrids. In immunization with thymus-dependent antigen, there is also the possibility of increasing the yield by adding some type of immune response modifier, so-called BRM (Biological Response Modifier). In so-called thymus-independent antigen, the direct yield is usually lower, and the BRMs that work well for thymus-dependent antigen do not work as well for thymus-independent antigen.

In an attempt to avoid these problems, so-called polyclonal activators, eg endotoxins, lectins, etc., have been added during the immunization. The disadvantage of this method, however, is that these activators stimulate all cells in the lymphocyte preparation, ie also the cells that have a suppressive effect on the immunization.

For stimulation of human lymphocytes, Borrebaeck et al (WO88 / 01642) have developed a system in which unwanted cells for in vitro immunization are removed, thus increasing the specific yield of antibody-producing hybridomas. The method is based on the use of a lysosomotropic agent that kills all lysosome-containing cells.

It has now surprisingly been found that by using a combination of the method with lysosomotropic agents and stimulation with polyclonal activators, the yield of specific hybridomas and also of specific IgG-producing hybridomas can be increased by in vitro immunization with both thymus-dependent and thymus-independent antigens. Thus, an object of the present invention is to provide a method for increasing the yield of antigen-positive hybridomas and increasing the yield of IgG-producing hybridomas of antigen-positive hybridomas in in vitro immunization of lymphocyte-containing cell populations to produce monoclonal antibodies.

The method according to the invention is characterized in that lysosomotropic agents, lysosomotropic derivatives thereof or lysosomotropic substances synthesized from such agents are caused to act in vitro on the lymphocyte-containing cell populations to remove cell populations with a negative effect on the in vitro immunosoculation, thymus-dependent or thymus-independent antigen in the simultaneous presence of a polyclonal activator in a suboptimal amount.

This method is thus based on the removal of cells that are undesirable for the immunization by means of a lysosomotropic agent, after which the remaining cells are utilized in a traditional in vitro immunization procedure while being stimulated with a polyclonal activator. The concentration of the polyclonal activator is such that the polyclonal activator itself is not able to stimulate the cells to proliferate, ie a suboptimal amount.

The cells immunized in this way are then fused in the traditional manner.

As polyclonal activators, for example, endotoxins can be used; lectins such as PHA (Phytohaemagglutinin), PWM (Pokeweed Mitogen), Con A (Concanavalin A); Staphylococcus aureus cells; protein A or protein G.

Lysosomotropic agents can be used as lysosomotropic amino acid derivatives or peptides based on such derivatives. An especially preferred lysosomotropic agent is leucine-leucine-O-methyl ester or peptides based on leucine-O-methyl ester. 461 10 15 20 25 30 35 985 4 These lysosomotropic agents have the specific ability to kill all lysosome-containing cells, eg monocytes / macrophages, NK cells and possibly other currently unknown cell subpopulations, in a very short time. Thus, by the use of lysosomotropic agents, a lymphocyte population can be adapted in a few tens of minutes so that it can be used in in vitro immunization for the production of monoclonal antibodies. Thus, the B cells of the lymphocyte population can be activated antigen-specifically without adverse effects from lysosomal-positive cells. In this way, an immune population of lymphocytes is created that can be used for the production of hybridomas and for the production of monoclonal antibodies.

The method can be applied to cell populations of animal origin. For example, experiments performed on murine lymphocyte-containing cell populations have given very good results.

Another object of the invention is to provide a kit of material, so-called kit, for use in in vitro immunization of lymphocyte-containing cell populations. The cell populations treated with this kit can then be used to produce monoclonal antibodies.

The kit according to the invention comprises at least three containers, of which one container contains lymphokines as active ingredient, one container contains lysosomotropic agent, lysosomotropic derivatives thereof or lysosomotropic substances synthesized from such agent as active ingredient and one container contains polyclonal activator as active ingredient. The kit also contains various disposable materials to facilitate the effect of the reagent on the cell populations, as well as a description of how it is to be used.

The invention is described in more detail in the following exemplary embodiments. 10 15 20 25 30 35 461 985 Example 1 læmuaiseria9_ @ eê_ëz @ 9§ëer9eaë§_§§§i9§n_9tën_§i11§§ & § ëy_e9lyë19a§l_§ë§¿y§§9: Musmjältceller (10 X 106 i DMEM with 10% fetal calf serum (FCS) (D10) was treated with 1eucine-1eucine-O-methyl ester, 25 pM, for 15 min in cells / ml) suspended room temperature. The cells were washed 2-3 times in DMEM with 2% FCS (D2). They were then subjected to an in vitro immunization with the thymus-dependent antigen conalbumin, 1 μg / ml, as immunogen. The culture also contained MLC (supernatant from mixed lymphocyte culture) and supernatant from stimulated EL-4 cells (Glad, C., Wennerström, G. and Fredlund, B.-M. in IN VITRO IMMUNIZATION IN HYBRIDOMA TECHNOLOGY (Borrebaeck , CAK Ed.) (1987), pp. 105-113, Elsevier Science Publishers). After 5 days of immunization, the cells were fused with myeloma cells from the Sp2 / 0 cell line. After 12-14 days, the growing hybridomas were tested for the production of antigen-specific antibodies and the production of IgG subclass antibodies, respectively.

Non-leucine-leucine-O-methyl-treated cells were used as a control.

The results are shown in Table 1.

Table 1 Cells% antigen-positive% IgG-producing hybridoma hybridoma of antigen-positive hybridoma untreated mouse spleen cells 5.3 14.3 1eu-leufQ-met-treated mouse spleen cells 5.3 42.9 Example 2 šmæ2§i§e§in9_meë_Ez @ 2§äer9eaëe_§e§i9sa_me§_§i1l§e§§ ë! _E91zkl9§§1_§k§i2§29r Mouse spleen cells were treated as in Example 1 with leucine-1eucine-0-methyl ester. In the subsequent immunization, conalbumin was used as the immunogen (1 pg / ml) with the addition of pokeweed mitogen (PWM) as the polyclonal activator at a final concentration of 0.1%. The immunization was allowed to proceed for 5 days, after which the cells were fused as in Example 1.

Cells not treated with leucine-leucine-O- -methyl or PWM were used as controls.

The results are shown in Table 2.

Table 2 Cells% antigen-positive% IgG-producing hybridoma hybridoma of antigen-positive hybridoma untreated mouse spleen cells 5.3 14.3 leu-leu-O-met and PWM-treated mouse spleen cells 31.9 62.4 Example 3 ___________ ______ _________________ ________________ §_29lyël92al_§ë§¿2§E9z Mouse spleen cells were treated as in Example 1 with leucine-leucine O-methyl ester. In the subsequent immunization, the thymus-independent antigen dextran, 1 pg / ml, was used. The immunization was allowed to proceed for 5 days, after which the cells were fused as in Example 1.

Non-leucine-1eucine-O-methyltreated cells were used as a control.

The results are shown in Table 3.

Table 3 Cells% antigen-positive% IgG-producing hybridoma hybridoma of antigen-positive hybridoma untreated mouse spleen cells 3.5 0 leu-leu-O-met-treated mouse spleen cells 4.5 0 10 15 20 25 30 461 985 Exemgel 4 læmuniss§i §¶_ @ së_§z @ 9§9ës: 9s§ës_맧i9sa_ @ së_ëillästs §y_p9lzäl9n§l_ëë§iyë§9: The experiment according to example 2 was repeated, but as an immunogen the thymus-independent antigen dextran, 1 pg / ml, was used with addition of pokeweed mitogen (PWM) as polyclonal activator.

Cells not treated with leucine-leucine-O-methyl or PWM were used as controls.

The results are shown in Table 4.

Table 4 Cells% antigen-positive% IgG-producing hybridoma hybridoma of antigen-positive hybridoma untreated mouse spleen cells 3.5 leu-leu-O-met and PWM-treated mouse spleen cells 27.7 10.6 From the experiments described above it appears that obtaining an increase in both the yield of antigen-positive hybridomas and in the proportion of IgG-producing hybridomas of the antigen-positive hybridomas when using a combination of either a thymus-dependent or a thymus-independent antigen and a polyclonal activator, when comparing untreated spleen cells and leu leu-O-met-treated and PWM-treated mouse spleen cells.

Claims (8)

461 10 15 20 25 30 985 PATENT REQUIREMENTS 1. A method of increasing the yield of antigen-positive hybridomas and increasing the yield of IgG-producing hybridomas of antigen-positive hybridomas in in vitro immunization of lymphocyte-containing cell populations to produce monoclonal antibodies, characterized in that lysosomotropic agent , lysosomotropic derivatives thereof or lysosomotropic substances synthesized from such an agent are caused to act in vitro on the lymphocyte-containing cell populations to remove cell populations adversely affecting the in vitro immunization, after which the lymphocytes are immunized in vitro with thymus-dependent or a polyclonal activator in a suboptimal amount. A method according to claim 1, characterized in that the polyclonal activator is selected from endotoxins, lectins, Staphylococcus aureus cells, protein A or protein G. 3. A method according to claim 1, characterized in that lysosomotropic amino acid derivatives or peptides based on such derivatives are used as lysosomotropic agents. Process according to Claim 1, characterized in that leucine-leucine-O-methyl ester or peptides based on leucine-O-methyl ester are used as lysosomotropic agent. Kit for use in immunizing lymphocyte-containing cell populations for the production of monoclonal antibodies, characterized in that it comprises at least three containers, one of which contains lymphokines as active ingredient, one container containing lysosomotropic agent, lysosomotropic derivatives thereof or lysosomotropic substances synthesized from such an agent as the active ingredient and a container containing a polyclonal activator in a suboptimal amount as an active ingredient. The kit of material according to claim 5, characterized in that the polyclonal activator is selected from endotoxins, lectins, Staphylococcus aureus cells, protein A or protein G. A kit of material according to claim 5 or 6, characterized in that the lysosomotropic agent is lysosomotropic amino acid derivatives or peptides based on such derivatives. 8. A kit of material according to claim 5 or 6, wherein leucine-leucine-O-methyl ester or peptides are based on the ability of the lysosomotropic agent to be on leucine-O-methyl ester.