SI22385A - Method for determination of proportion and quality of hybridoms and application of hybridoms of appropriate quality - Google Patents

Abstract

The present invention relates to a method for determining the ratio and quality of hybridomas by means of the number of differently labeled lysosomes, usually labeled with green and red fluorescence, where lysosomes originate from eukaryotic parent cells. Due to the property of lysosomes of fusing with one another, also yellow lysosomes formed by the fusion of green and red lysosomes may be found in the population of said green and red lysosomes. The physiological process of lysosomal fusion is one of the events involved in a complex system of expressing tumour and other antigens on the hybridoma surface. Therefore this new method gives, in addition to the data on the amount of hybridomas in a suspension of fused cells, at the same time also indirect information on the immunogenicity of hybridomas, which is of key importance for the effectiveness of a cellular vaccine against cancer in the paticnt's body.

Description

FIELD OF THE INVENTION

The present invention is in the field of cell biology, therapeutic biotechnology and immunology and relates to a method for determining the proportion and quality of hybridomas resulting from fusion of eukaryotic cells, e.g. dendritic and tumor cells. In the following, we will focus on the fusion between dendritic and tumor cells.

The subject of the invention is a method of confocal microscopy for determining the proportion of hybridomas by means of fluorescently labeled subcellular organelles, e.g. lysosomes. At the same time, the method can also determine the proportion of fused lysosomes in a single hybridoma. This information is very important for determining the quality of the cell vaccine. Indeed, dendritic and tumor cell hybridomas are cellular vaccines for the treatment of cancer, and potentially, hybridomas prepared by this method can also be used generally to modulate the immune response.

Determining the proportion and quality of hybridomas is a very important step in their production.

According to the new method, the proportion of hybridomas is determined by confocal microscopy by detecting lysosomes and by mediating the fusion of lysosomes derived from different parental cells, which may be of human and / or animal origin. The process of lysosome fusion is the cornerstone of antigen presentation in antigen presenting cells. Therefore, even in dendritic and tumor cell hybridomas, lysosome fusion is directly involved in the antigen presentation pathway. Tumor peptides from tumor cell lysosomes bind to molecules of major histocompatible system (PHK) class II derived from dendritic cell lysosomes. So we take advantage of the property of lysosomes to fuse with each other and connect as above, and only in this case all known and unknown tumor antigens are presented on the cells. This, however, is a prerequisite for the quality of hybridomas suitable for the production of the cell vaccine.

The method of the invention is also useful for labeling and determining the number of antibody-synthesizing hybridomas and for other applications of subcellular structure dynamics studies.

A technical problem

So far, the determination of the proportion of hybridomas has been based on the standard flow cytometry method, which allows the measurement of yellow double-fluorescence cells, whose cytoplasm contains a mixture of green and red fluorescence cytoplasmic markers, which are separately introduced into the parent cells. However, due to technical limitations, flow cytometry also detects cell clusters as hybridomas, so it is better to identify hybridomas by confocal microscopy (Gabriel et al., Quantification of whole hybridoma yields with confocal microscopy and flow cytometry, Biochem. Biophys. Res. Commun., Vol 314, 2004, Volume 3, Pages 717-723). In both cases, however, the results obtained relate solely to the amount of fusion product. It also turned out that the method described is not sensitive enough to determine the quality of individual hybridomas. In addition, some scientists have determined the number of hybridomas in fused samples after incubated incubation at 37 ° C (Orentas et al., Electrofusion of a weakly immunogenic neuroblastoma with dendritic cell produces a tumor vaccine, Celi Immunol., Vol. 213, 2001). Volume 1, Pages 4-13). According to the literature, this is not sufficient for the preparation of biologically effective cell vaccines, since the course of antigen presentation is much slower (Pierre et al., Developmental regulation of MHC class II transport in mouse dendritic cellulose, Nature, vol. 388, 1997 6644, pages 787-792; Roosnek E et al., Kinetics of MHC-antigen complex formation on antigen-presenting cellulose, J. Immunol., Vol. 140, 1988, Volume 12, pages 4079-4082). Thus, not enough tumor antigens in complexes with PHK molecules are exposed on the surface of hybridomas. The use of such hybridomas thus cannot induce maximal T cell activation against the full spectrum of tumor antigens, that is, known and unknown tumor antigens.

So ideally, the proportion of hybridomas could be determined using a strategy that would allow for the detection of a more biologically relevant process, e.g. such as antigen presentation.

The state of the art

The advantage of cell hybridomas over other cellular vaccines is that, through highly specialized antigen-presenting mechanisms of dendritic cells, they present on their surface both known and unknown tumor antigens in this form (Gong et al., Induction of antitumor activity by immunization with fusions of dendritic and carcinoma celiš, Nat. Med., vol. 3, 1997, pages 558 - 561) to be recognized by lymphocytes T. Research in the field of cell hybridomas has progressed to clinical trials where immune cells have otherwise been shown to activate. and only a limited group of people show clinical effects. The reasons for this can be attributed in part to the inappropriate method for preparing and determining the proportion of hybridomas by flow cytometry. The amount of hybridomas as well as the proper presentation of tumor antigens are crucial for the activation of the immune response. Today, the proportion of hybridomas is determined by flow cytometry by staining the same amount of partner cells with green or green cells. red fluorescence cytoplasmic dyes (Jaroszeski et al., Flow cytometric detection and quantitation of cell-cell electrofusion products, Methods Mol. Biol., vol. 91, 1998, pages 149-156). The number of hybridomas is determined after electrofusion by searching for double-fluorescence cells, which are colored yellow due to mixing of cytoplasmic markers. However, over time, this technique has proven to be less sensitive (Hayashi et al., Immunogenicity and therapeutic efficacy of dendritic-tumor hybrid whole cell generated by electrofusion, Ciin. Immunol., Vol. 104, pages 14-20) in determining the quality of individual cells. In addition, yellow staining of cells occurs shortly after fusion, which is why some scientists determine their number in fused samples after incubating at 37 ° C (Orentas et al., Electrofusion of a weakly immunogenic neuroblastoma with dendritic cell produces a tumor vaccine, Celi Immunol vol. 213, 2001, vol. 1, pages 4-13). At this time, however, there is likely to be no effective exposure of tumor antigens to the membrane of the hybridoma. Thus prepared hybridomas do not elicit mass activation of relevant immune cells against the full spectrum of known and unknown tumor antigens. This may also be one of the causes of the adverse effect of the vaccine reported in some studies (Orentas et al., Electrofusion of a weakly immunogenic neuroblastoma with dendritic cellulitis produces a tumor vaccine, Celi Immunol vol. 213, 2001, Volume 1, pages 4-13).

The solution to a technical problem

These disadvantages can be successfully remedied by the method of the invention. We control the average proportion of hybridomas by varying the extent of lysosome fusion using confocal microscopy. This method also allows the distinguishing of lysosomes and allows the determination of their fusion status within hybridomas, which flow cytometry cannot detect. Lysosomes are dynamic, fusible organelles that are involved in the presentation of antigens. Hybridomas are identified using differentially labeled lysosomes (usually green and red) that are directly involved in the antigen expression process in the hybridomas. Among the lysosomes from both types of parental cells, the physiological process of fusion of lysosomes can be observed under certain incubation conditions - time and temperature. The fusion of the lysosomes of both parent cells is part of the antigen presentation pathway in the hybridomas and allows the association of tumor peptides located in the tumor cell lysosomes with molecules of the major histocompatible system derived from the dendritic cell lysosomes.

This approach provides a new method for optimizing the preparation of hybridoma cell vaccines based on the intracellular antigen presentation process.

This method covers:

- labeling of cell compartments, in the narrow sense, these are lysosomes involved in the expression of antigens;

- determination of the number of lysosomes in a single cell by confocal microscopy;

- determining the proportion of hybridomas based on the number of lysosomes derived from parental cells;

determining the proportion of fused lysosomes in the hybridoma under different incubation conditions using e.g. confocal microscopy. In addition, this method also provides us with information on the activity of the antigen presentation pathway in the hybridoma, which depends on the biological efficacy of the cellular vaccine.

The first object of the invention is therefore a method for determining the proportion and quality of eukaryotic cell hybridomas, characterized by the labeling of lysosomes of parental cells of human and / or animal origin, wherein one type of parental cells may be tumor cells labeled with one color, and they are another type of parental cells of another eukaryotic cell, preferably dendritic cells labeled with a different color, the cells are fused and the extent of lysosome fusion measured.

More specifically, the above method is characterized by the fact that,

- Fluorescently label the lysosomes

- by means of confocal microscopy, in a known manner, the minimum number of lysosomes in individual parent cells is determined,

- individual parental cells are fused and incubated after fusion for 2 to 30 hours at a temperature of 20 to 37 ° C,

- Potential hybridomas are identified in a known manner using confocal microscopy;

- determine the number of hybridomas as the number of cells with a minimum number of differently stained unbound lysosomes from both parent cells and a minimum number of fused lysosomes identified as otherwise stained,

- express the proportion of hybridomas as the ratio between the number of hybridomas and the number of all fluorescently labeled cells.

The new method of the invention differs from the classical method for determining the number of hybridomas in that we do not label cytoplasm but lysosomes that are directly involved in the process of presenting tumor antigens. The number of lysosomes within hybridomas can only be determined by confocal microscopy. Thus, the new method by confocal microscopy differs from the flow cytometry method used so far in that it not only provides accurate information on the quantity of hybridomas, but is also suitable for determining the quality of individual cells, ie the quality of hybridomas in suspension of the cell vaccine.

So far, no method has been available to allow the determination of the proportion of hybridomas by cellular compartments directly involved in the antigen presentation pathway of hybridomas.

The method of the invention exploits the property of lysosomes to merge with one another. Cell lysosomes of different origin also merge with each other.

Fluorescently labeled molecules of dextrans or other similar molecules are used to label the lysosomes, which are incorporated into the cell by the endocytotic pathway. For each of the two types of parental cells, e.g. dendritic and tumor cells, we use a different fluorescent dye, we usually use green and red fluorescently labeled cells, but we can also use other dyes. The journey of dextran to lysosomes lasts from a few to at least 20 hours and more at 37 ° C, depending on the cell type. Dextran molecules are removed from early and late endosomal compartments by non-ciliary incubation at

37 ° C in nutrient medium in the absence of dextran to avoid disturbing quantification measurements in lysosomes.

This method is the basis both for determining the amount of hybridomas in the cell vaccine and for determining the activity of the antigen presentation pathway in hybridomas under different incubation conditions.

The method of the invention comprises the following sequential steps:

a) in a known manner, determining the minimum number of labeled lysosomes in each parent cell;

b) capturing confocal images of fused and control samples, that is, samples of cells that were not exposed to the electro-fusion process;

c) capture consecutive vertical optical planes using confocal microscopy for each presumed hybrid;

d) determining the number of non-fused lysosomes from each parent cell individually and determining the number of fused lysosomes;

e) determining the number of hybridomas in each sample on the basis of the minimum number of green and red lysosomes, that is, on the basis of the minimum number of lysosomes from both parental cells, and the minimum number of yellow lysosomes, that is, fused lysosomes, where in step a) confocal microscopy successive vertical optical planes for a few randomly selected unfused, dextran-labeled cells. The number of lysosomes per cell is determined by counting all lysosomes on each optical plane of the selected cell, taking into account that the image of each lysosome can be located in up to three optical planes. The counting process is performed separately for each cell type. After the counting process is completed, a minimum number of lysosomes per cell is determined.

in step b) a sample of cells after fusion and from 2 to 30 µm incubation at a temperature of 20 to 37 ° C is analyzed by confocal images captured by a confocal microscope, with green fluorescence evoked by argon hair and red fluorescence by helium neon lasers;

in step c), in the fused cell sample, potential hybridomas are identified in a known manner and sequential vertical optical planes for each hypothesized hybrid are captured by confocal microscopy;

in step d), using a computer program, determine the number of green, red, and yellow (fused) lysosomes, and, based on the number of red, green, and yellow lysosomes, determine which cells are hybridomas. On this basis, we can also determine whether hybrids are of adequate quality, that is, with as many fused lysosomes as possible;

in step e), the hybridomas in the sample are counted, and then their proportion is expressed as the ratio between the number of hybridomas and the number of all fluorescently labeled cells.

Another object of the invention is to use hybridomas obtained by the method of the invention with as many fused lysosomes as possible for the preparation of a cellular cancer vaccine.

As is known, the tumor cells of a patient to be vaccinated are used to prepare a cancer cell vaccine as one type of parental cell. These cells could be treated by the method of the invention and the hybridomas obtained and selected as indicated above would be used to prepare a cell vaccine to vaccinate this patient.

A third object of the invention is the use of hybridomas obtained by the method of the invention and selected as above for modulating the immune response.

The method of the invention is explained in more detail in Figures 1 and 2.

Figure 1 shows a method for determining the number of lysosomes by sequential image acquisition in confocal optical planes by confocal microscopy. When counting lysosomes, we considered the possibility that each lysosome could occur in more than one plane. Figure 1 a represents a sequence of vertical optical planes. The thickness of the optical plane is 0.5 pm. The white circle in each plane indicates the lysosome. Mark A.E. means an arbitrary unit. Figure 1 b shows a sequence of line diagrams showing the fluorescence intensity of the respective lysosome on the left side of Figure 1 a, and confirms our assumption that the lysosome image can also extend across three optical planes.

Figure 2 a represents a sample of cells exposed to a high voltage electric shock. The image was captured by the screening light; green and red areas, however, are dextran-labeled lysosomes. After electrofusion, the cells in the image were incubated for 20 hours at 37 ° C. The frame is surrounded by hybrids containing at least a minimum number of green and red or at least a minimum number of yellow lysosomes and two nuclei (J).

Confocal Figure 2 b is a magnification of the hybridoma in the frame of Figure 2 a. The yellow areas represent the fused (green and red) lysosomes in the hybridoma. The average overlap of green and red areas in this figure is 75%.

The bottom right corner of Figures 2 a and 2 b represent the 20 pm scale.

The invention illustrates the following example.

Example

Both samples of parental cells (dendritic and tumor) were incubated separately before electrofusion in the presence of green or red dextran typically 4 to 20 hours or more. After incubation, the cells were washed and incubated for an additional few hours in nutrient medium in the absence of dextran. The average number of lysosomes in a single cell is determined by counting the lysosomes with a computer program in each of the recorded (confocal microscopy) sequential vertical optical planes of each unfused cell (Figure 1 a). In doing so, we consider that each lysosome can be located in up to three optical planes (Figure 1 b). Equal amounts of cells with green or red labeled lysosomes are exposed to the fusion procedure in an electric field and then incubated for 20 hours at 37 ° C. Samples were then analyzed using images taken with confocal microscopy (Figures 2 a, 2 b). Confocal microscopy is used to look for potential hybridomas, cells with a minimal number of green, red and yellow lysosomes. Potential hybridomas are then analyzed using recorded sequential vertical optical planes by counting the number of green, red, and yellow lysosomes in the hybrid using a computer program. The proportion of yellow, fused lysosomes in the hybridoma is then expressed as the ratio of the number of yellow pixels to the number of all fluorescence pixels on each optical plane of each hybridoma. We then determine the proportion of all hybridomas in the sample, expressing it as the relationship between the number of hybridomas and the number of all fluorescently labeled cells in the confocal images.

Claims (5)

Patent claims 1. A method for determining the proportion and quality of eukaryotic cell hybridomas by confocal microscopy, characterized by the labeling of lysosomes of parental cells of human and / or animal origin, wherein one type of parental cells may be tumor cells characterized by one color and are another type of parental cells of another eukaryotic cell, preferably dendritic cells labeled with a different color, the cells fuse and measure the extent of lysosome fusion. Method according to claim 1, characterized in that - Fluorescently label the lysosomes - by means of confocal microscopy, in a known manner, the minimum number of lysosomes in individual parent cells is determined, - individual parental cells are fused and incubated after fusion for 2 to 30 hours at a temperature of 20 to 37 ° C, - Potential hybridomas are identified in a known manner using confocal microscopy; - determine the number of hybridomas as the number of cells with a minimum number of differently stained unbound lysosomes from both parent cells and a minimum number of fused lysosomes identified as otherwise stained, - express the proportion of hybridomas as the ratio between the number of hybridomas and the number of all fluorescently labeled cells. A method according to claim 1 or 2, characterized in that the lysosomes are labeled with fluorescently labeled dextran molecules. Use of hybridomas obtained by the method of claim 1 with as many fused lysosomes as possible for the preparation of a cellular cancer vaccine. Use of hybridomas obtained by the method of claim 1 with as many fused lysosomes as possible to modulate the immune response.