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WO1990002795A1 - In vitro immunisation of lymphocyte-containing cell populations and kit therefor - Google Patents

In vitro immunisation of lymphocyte-containing cell populations and kit therefor Download PDF

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Publication number
WO1990002795A1
WO1990002795A1 PCT/SE1989/000486 SE8900486W WO9002795A1 WO 1990002795 A1 WO1990002795 A1 WO 1990002795A1 SE 8900486 W SE8900486 W SE 8900486W WO 9002795 A1 WO9002795 A1 WO 9002795A1
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Prior art keywords
vitro
cell populations
hybridomae
lymphocyte
kit
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PCT/SE1989/000486
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French (fr)
Inventor
Cristina Glad
Carl Borrebaeck
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Bioinvent International Ab
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Publication of WO1990002795A1 publication Critical patent/WO1990002795A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention comprises a method for increas ⁇ ing the yield of antigen-positive hybridomae and the yield of IgG-producing hybridomae amongst antigen-positive hy ⁇ bridomae in in vitro immunisation of lymphocyte-containing cell populations, and a kit for use in such in vitro immu- nisation.
  • Monoclonal antibodies were introduced in 1975 by K ⁇ hler and Milstein. The concept implies fusing immune lymphocytes with a continuous cell line, for example myeloma. A cloning and selection method makes it possible to select and culture cells producing a specific antibody. This cell clone then originates from one original cell (“monoclonal”) and produces exactly identical copies of a specific antibody.
  • the immune lymphocytes used in the method according to K ⁇ hler and Milstein were mouse lymphocytes that had been immunised in vivo.
  • Immunisation itself has also developed and may now be carried out in vitro, i.e. in cell culture environment.
  • antigens can be used, including antigen which are toxic or pathogenic for mice.
  • These in vitro im ⁇ munised lymphocytes are fused in the same manner as lympho cytes which have been immunised in vivo.
  • This technique ha been developed over a number of years, and there are now well-developed in vitro immunisation systems.
  • a characteristic feature of these systems is, how ⁇ ever, that they chiefly result in a primary immunisation with a production of IgM-type antibodies.
  • Different methods have been tested to achieve a transition from IgM production to IgG production in an in vitro system, which in vivo occurs naturally for most antigens.
  • the present in vitro immunisation technique provides a satisfactory yield of specific hybri ⁇ domae.
  • BRM Biological Response Modifier
  • thymus-independent antigens the direct yield is mostly lower, and BRMs functioning well with thymus-depen- dent antigens do not function as well with thymus-indepen ⁇ dent ones.
  • polyclonal activators e.g. endotoxins or lec- tins
  • the disadvantage of this method being that the activators stimulate all cells in the lym ⁇ phocyte preparation, including the cells which have a sup- pressive effect on the immunisation.
  • Borrebaeck et al ( O88/01642) have developed a syste for the stimulation of human lymphocytes, in which cells not wanted for the in vitro immunisation are removed, so as to increase the specific yield of antibody-producing hybridomae.
  • the method is based on the use of a lysosomo ⁇ tropic agent killing all lysosome-containing cells.
  • one object of the present invention is to pro ⁇ vide a method for increasing the yield of antigen-positive hybridomae and the yield of IgG-producing hybridomae amongst antigen-positive hydridomae in in vitro immunisa ⁇ tion of lymphocyte-containing cell populations.
  • a method according to the invention is characterised in that lysosomotropic agents, derivatives thereof, or substances synthetised on the basis of these agents are caused to act in vitro on the lymphocyte-containing cell populations for the removal of cell populations having a negative effect on the in vitro immunisation, whereupon the lymphocytes are in vitro immunised with thymus-depen- dent or thymus-independent antigens in the presence of a polyclonal activator.
  • Said method is thus based on cells unwanted for the immunisation being removed with the aid of a lysosomotro ⁇ pic agent, whereupon the remaining cells are used in a traditional in vitro immunisation method, while stimulated with a polyclonal activator.
  • concentration of the polyclonal activator is such that the polyclonal activator in itself is not capable of stimulating the cells to proliferation, i.e a suboptimal quantity.
  • the cells thus immunised are thereafter fused in tra ⁇ ditional manner.
  • endotoxins such as PHA (Phyto- haemagglutinin), PWM (Pokeweed Mitogen), Con A (Concanava- lin A); Staphylococcus aureus cells; protein A or protein G may be used as polyclonal activators.
  • PHA Physical- haemagglutinin
  • PWM Pokeweed Mitogen
  • Con A Concanava- lin A
  • Staphylococcus aureus cells protein A or protein G
  • protein A or protein G may be used as polyclonal activators.
  • Lysosomotropic amino acid derivatives or peptides based on such derivatives may be used as lysosomotropic agents.
  • a specially preferred lysosomotropic agent is leu- cine-leucine-O-methyl ester, or peptides based on leucine- O-methyl ester. These lysosomotropic agents have the specific ability to kill all lysosome-containing cells, for example mono- cytes/macrophages, NK cells and, possibly, other presently unknown cell subpopulations, in a very short time.
  • lymphocytes By using lysosomotropic agents, one may, in a few dozen mi ⁇ nutes, adapt a lymphocyte population so that it can be used in in vitro immunisation for production of monoclonal antibodies.
  • the B cells of the lymphocyte population may thus be antigen-specifically activated without any nega- tive effect from lysosome-positive cells. In this manner, one creates an immune population of lymphocytes that can be used for producing hybridomae and monoclonal antibo ⁇ dies.
  • the method can be applied to cell populations of ani- mal origin, and excellent results have, for example, been obtained in tests involving murine lymphocyte-containing cell populations.
  • Another object of the invention is to provide a metho for increasing the yield of IgG-producing hybridomae amongst antigen-positive hybridomae in in vitro immunisa ⁇ tion of lymphocyte-containing cell populations, said metho being characterised in that the lymphocytes are in vitro immunised with thymus-dependent or thymus-independent anti gens in the presence of a polyclonal activator.
  • a further object of the invention is to provide a kit for use in in vitro immunisation of lymphocyte-containing cell populations. The cell populations treated by means of this kit can thereafter be used for producing monoclonal antibodies.
  • the kit according to the invention comprises at least three containers, of which one container holds lymphokines as active constituent, one container holds lysosomotropic agents, derivatives thereof, or substances synthetised on the basis of such an agent as active constituent, and one container holds a polyclonal activator as active consti ⁇ tuent.
  • the kit also contains various disposable materials to improve the effect of the reactants on the cell popula tions, as well as instructions how to use said kit.
  • kits according to the invention has no con ⁇ tainer with lysosomotropic agents but is otherwise identi- cal with the above-mentioned kit.
  • Example 1 [mmuni ation_with a ;th ⁇ mus_ ⁇ dependent_anti.gen without addi ⁇ _t:Lon_ ⁇ j a £ ⁇ _ly ⁇ lonal_actLvator
  • Mouse spleen cells (10 x 10 cells/ml) suspended in DMEM with 10% foetal calf serum (FCS) (D10) were treated with leucine-leucine-O-methyl ester (25 ⁇ M) during 15 min. at room temperature. The cells were washed 2-3 times in DMEM with 2% FCS (D2), whereupon they were subjected to in vitro immunisation with the thymus-dependent antigen con- albumin (1 ⁇ g/ml) as immunogen. The culture also contained MLC (supernatant from mixed lymphocyte culture) and a su ⁇ pernatant from stimulated EL-4 cells (Glad, C, Wenner- str ⁇ m, G. and Fredlund, B.-M.
  • MLC supernatant from mixed lymphocyte culture
  • a su ⁇ pernatant from stimulated EL-4 cells Gad, C, Wenner- str ⁇ m, G. and Fredlund, B.-M.
  • Mouse spleen cells were treated with leucine-leucine- O-methyl ester, as in Example 1.
  • conalbumin was used as immunogen (1 ⁇ g/ l), poke- weed mitogen (PWM) being added as polyclonal activator in a final concentration of 0.1%.
  • PWM poke- weed mitogen
  • Mouse spleen cells were treated with leucine-leucine- O-methyl ester, as in Example 1.
  • the thymus-independent antigen dextran (1 ⁇ g/ml) was used. After 5 days of immunisation, the cells were fused, as in Example 1.
  • Example 2 The test according to Example 2 was repeated, but the thymus-independent antigen dextran (1 ⁇ g/ml) was used as immunogen, pokeweed mitogen (PWM) being added as polyclo ⁇ nal activator.
  • PWM pokeweed mitogen

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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

IN VITRO IMMUNISATION OF LYMPHOCYTE-CONTAINING CELL POPULATIONS AND KIT THEREFOR
The present invention comprises a method for increas¬ ing the yield of antigen-positive hybridomae and the yield of IgG-producing hybridomae amongst antigen-positive hy¬ bridomae in in vitro immunisation of lymphocyte-containing cell populations, and a kit for use in such in vitro immu- nisation.
Monoclonal antibodies were introduced in 1975 by Kδhler and Milstein. The concept implies fusing immune lymphocytes with a continuous cell line, for example myeloma. A cloning and selection method makes it possible to select and culture cells producing a specific antibody. This cell clone then originates from one original cell ("monoclonal") and produces exactly identical copies of a specific antibody.
The immune lymphocytes used in the method according to Kδhler and Milstein were mouse lymphocytes that had been immunised in vivo.
In this manner, monoclonal antibodies have been pre¬ pared against a number of different molecules, and this type of antibody has been used, and is still being used, to an immense extent. Also, the commercial development has been considerable, and today a large number of monoclonal antibodies are being marketed, above all for diagnostic purposes.
Immunisation itself has also developed and may now be carried out in vitro, i.e. in cell culture environment.
Thus, all types of antigens can be used, including antigen which are toxic or pathogenic for mice. These in vitro im¬ munised lymphocytes are fused in the same manner as lympho cytes which have been immunised in vivo. This technique ha been developed over a number of years, and there are now well-developed in vitro immunisation systems. A characteristic feature of these systems is, how¬ ever, that they chiefly result in a primary immunisation with a production of IgM-type antibodies. Different methods have been tested to achieve a transition from IgM production to IgG production in an in vitro system, which in vivo occurs naturally for most antigens.
For most antigens, the present in vitro immunisation technique provides a satisfactory yield of specific hybri¬ domae. In immunisation with thymus-dependent antigens, there is furthermore the possibility of increasing the yield by adding some type of immune response modifying agent, so-called BRM (Biological Response Modifier). In so-called thymus-independent antigens, the direct yield is mostly lower, and BRMs functioning well with thymus-depen- dent antigens do not function as well with thymus-indepen¬ dent ones.
In an attempt to avoid these problems, one has added so-called polyclonal activators, e.g. endotoxins or lec- tins, in the immunisation, the disadvantage of this method being that the activators stimulate all cells in the lym¬ phocyte preparation, including the cells which have a sup- pressive effect on the immunisation.
Borrebaeck et al ( O88/01642) have developed a syste for the stimulation of human lymphocytes, in which cells not wanted for the in vitro immunisation are removed, so as to increase the specific yield of antibody-producing hybridomae. The method is based on the use of a lysosomo¬ tropic agent killing all lysosome-containing cells.
It has now surprisingly been' found that, by combinin the use of lysosomotropic agents and the stimulation with polyclonal activators, it is possible to increase the yield of specific hybridomae and also of specific IgG-pro ducing hybridomae, in in vitro immunisation with thymus- dependent, as well as thymus-independent, antigens. If the treatment with lysosomotropic agents is left out, one obtains an increase only in the yield of spe- cific IgG-producing hybridomae, when using a well-adjusted quantity of polyclonal activators.
Thus, one object of the present invention is to pro¬ vide a method for increasing the yield of antigen-positive hybridomae and the yield of IgG-producing hybridomae amongst antigen-positive hydridomae in in vitro immunisa¬ tion of lymphocyte-containing cell populations.
A method according to the invention is characterised in that lysosomotropic agents, derivatives thereof, or substances synthetised on the basis of these agents are caused to act in vitro on the lymphocyte-containing cell populations for the removal of cell populations having a negative effect on the in vitro immunisation, whereupon the lymphocytes are in vitro immunised with thymus-depen- dent or thymus-independent antigens in the presence of a polyclonal activator.
Said method is thus based on cells unwanted for the immunisation being removed with the aid of a lysosomotro¬ pic agent, whereupon the remaining cells are used in a traditional in vitro immunisation method, while stimulated with a polyclonal activator. Possibly, the concentration of the polyclonal activator is such that the polyclonal activator in itself is not capable of stimulating the cells to proliferation, i.e a suboptimal quantity. The cells thus immunised are thereafter fused in tra¬ ditional manner.
For example endotoxins; lectins, such as PHA (Phyto- haemagglutinin), PWM (Pokeweed Mitogen), Con A (Concanava- lin A); Staphylococcus aureus cells; protein A or protein G may be used as polyclonal activators.
Lysosomotropic amino acid derivatives or peptides based on such derivatives may be used as lysosomotropic agents. A specially preferred lysosomotropic agent is leu- cine-leucine-O-methyl ester, or peptides based on leucine- O-methyl ester. These lysosomotropic agents have the specific ability to kill all lysosome-containing cells, for example mono- cytes/macrophages, NK cells and, possibly, other presently unknown cell subpopulations, in a very short time. By using lysosomotropic agents, one may, in a few dozen mi¬ nutes, adapt a lymphocyte population so that it can be used in in vitro immunisation for production of monoclonal antibodies. The B cells of the lymphocyte population may thus be antigen-specifically activated without any nega- tive effect from lysosome-positive cells. In this manner, one creates an immune population of lymphocytes that can be used for producing hybridomae and monoclonal antibo¬ dies.
The method can be applied to cell populations of ani- mal origin, and excellent results have, for example, been obtained in tests involving murine lymphocyte-containing cell populations.
Another object of the invention is to provide a metho for increasing the yield of IgG-producing hybridomae amongst antigen-positive hybridomae in in vitro immunisa¬ tion of lymphocyte-containing cell populations, said metho being characterised in that the lymphocytes are in vitro immunised with thymus-dependent or thymus-independent anti gens in the presence of a polyclonal activator. A further object of the invention is to provide a kit for use in in vitro immunisation of lymphocyte-containing cell populations. The cell populations treated by means of this kit can thereafter be used for producing monoclonal antibodies. The kit according to the invention comprises at least three containers, of which one container holds lymphokines as active constituent, one container holds lysosomotropic agents, derivatives thereof, or substances synthetised on the basis of such an agent as active constituent, and one container holds a polyclonal activator as active consti¬ tuent. The kit also contains various disposable materials to improve the effect of the reactants on the cell popula tions, as well as instructions how to use said kit.
Another kit according to the invention has no con¬ tainer with lysosomotropic agents but is otherwise identi- cal with the above-mentioned kit.
The invention will be described in detail in the fol¬ lowing Examples. Example 1 ][mmuni ation_with a ;thγmus_^dependent_anti.gen without addi^ _t:Lon_θj a £θ_lyσlonal_actLvator
Mouse spleen cells (10 x 10 cells/ml) suspended in DMEM with 10% foetal calf serum (FCS) (D10) were treated with leucine-leucine-O-methyl ester (25 μM) during 15 min. at room temperature. The cells were washed 2-3 times in DMEM with 2% FCS (D2), whereupon they were subjected to in vitro immunisation with the thymus-dependent antigen con- albumin (1 μg/ml) as immunogen. The culture also contained MLC (supernatant from mixed lymphocyte culture) and a su¬ pernatant from stimulated EL-4 cells (Glad, C, Wenner- strόm, G. and Fredlund, B.-M. in IN VITRO IMMUNISATION IN HYBRIDOMA TECHNOLOGY (Borrebaeck, C.A.K. Ed.) (1987), pp 105-113, Elsevier Science Publishers). After 5 days of immunisation, the cells were fused with myeloma cells fro the cell line Sp2/0 and, after 12-14 days, the growing hybridomae were tested as to the production of antigen- specific antibodies and the production of antibodies of Ig subclass, respectively.
Cells not treated with leucine-leucine-O-methyl were used for reference. The results are apparent from Table 1. Table 1
Cells % antigen-posi¬ % IgG-producing hyb tive hybridomae domae of the antige positive hybridomae
untreated mouse spleen cells 5.3 14.3 leu-leu-O-meth- treated mouse spleen cells 5.3 42.9
Example 2 ιmunisation_with a thyjnus^dependent_anti en_with addition of_a_pp_l] clpna_l aci:ivatoι:
Mouse spleen cells were treated with leucine-leucine- O-methyl ester, as in Example 1. In the following immuni¬ sation, conalbumin was used as immunogen (1 μg/ l), poke- weed mitogen (PWM) being added as polyclonal activator in a final concentration of 0.1%. After 5 days of immunisa¬ tion, the cells were fused, as in Example 1.
Cells not treated with leucine-leucine-O-methyl or PWM were used for reference.
The results are apparent from Table 2.
Table 2
Cells % antigen-posi¬ % IgG-producing hyb tive hybridomae domae of the antige positive hybridoma
untreated mouse spleen cells 5.3 14.3 leu-leu-O-meth- treated and PWM- treated mouse spleen cells 31.9 62.4 Example 3
Immuni^ation_w:Lth a th mus^independent_antigen_w^thout_any addition_of? a £θ3.yc^onal_actLvator
Mouse spleen cells were treated with leucine-leucine- O-methyl ester, as in Example 1. In the following immuni¬ sation, the thymus-independent antigen dextran (1 μg/ml) was used. After 5 days of immunisation, the cells were fused, as in Example 1.
Cells not treated with leucine-leucine-O-methyl were used for reference.
The results are apparent from Table 3.
Table 3
Cells % antigen-posi¬ % IgG-producing hyb tive hybridomae domae of the antige positive hybridomae
untreated mouse spleen cells 3.5 leu-leu-O-meth- treated mouse spleen cells 4.5
Example 4
Immunisation_with a th mus independent_antigen_with addi¬ tion of_a_pol clonaI. activator
The test according to Example 2 was repeated, but the thymus-independent antigen dextran (1 μg/ml) was used as immunogen, pokeweed mitogen (PWM) being added as polyclo¬ nal activator.
Cells not treated with leucine-leucine-O-methyl or PWM. were used for reference.
The results are apparent from Table 4. Table 4
Cells % antigen-posi¬ % IgG-producing hyb tive hybridomae domae of the antige positive hybridomae
untreated mouse spleen cells 3.5 leu-leu-O-meth- treated and PWM- treated mouse spleen cells 27.7 10.6
It is apparent from the tests described above that one obtains an increased yield of antigen-positive hybri- domae, as well as of the amount of IgG-producing hybri¬ domae amongst antigen-positive hybridomae, when using a combination of either a thymus-dependent or a thymus-inde¬ pendent antigen and a polyclonal activator, untreated mouse spleen cells being compared with mouse spleen cells treated with leucine-leucine-O-methyl and PWM.

Claims

1. A method for increasing the yield of antigen-po- sitive hybridomae and the yield of IgG-producing hybrido¬ mae amongst antigen-positive hybridomae in in vitro immu¬ nisation of lymphocyte-containing cell populations, c h a r a c t e r i s e d in that lysosomotropic agents, derivatives thereof, or substances synthetised on the basis of these agents are caused to act in vitro on the 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 immu¬ nised with thymus-dependent or thymus-independent antigens in the presence of a polyclonal activator.
2. A method for increasing the yield of IgG-producing hybridomae amongst antigen-positive hybridomae in in vitro immunisation of lymphocyte-containing cell populations, c h a r a c t e r i s e d in that the lymphocytes are in vitro immunised with thymus-dependent or thymus-indepen¬ dent antigens in the presence of a polyclonal activator.
3. A method as claimed in claim 1 or 2, c h a ¬ r a c t e r i s e d in that the polyclonal activator is chosen amongst endotoxins, lectins, Staphylococcus aureus cells, protein A or protein G.
4. A method as claimed in claim 1, 2 or 3, c h a ¬ r a c t e r i s e d in that the polyclonal activator is added in a suboptimal quantity.
5. A method as claimed in claim 1, c h a r a c - t e r i s e d in that the lysosomotropic agent employed consists of lysosomotropic amino acid derivatives or pep¬ tides based on such derivatives.
6. A method as claimed in claim 1, c h a r a c ¬ t e r i s e d in that the lysosomotropic agent employed is leucine-leucine-O-methyl ester, or peptides based on leucine-O-methyl ester. 10
7. A kit for use in immunisation of lymphocyte-con¬ taining cell populations, c h a r a c t e r i s e d in that it comprises at least three containers, of which one container holds lymphokins as active constituent, one con-
5 tainer holds lysosomotropic agents, derivatives thereof or substances synthetised on the basis of such an agent as active constituent, and one container holds a polyclonal activator as active constituent.
8. A kit for use in immunisation of lymphocyte-con- 10 taining cell populations, c h a r a c t e r i s e d in that it comprises at least two containers, of which one holds lymphocytes as active constituent and the other holds a polyclonal activator as active constituent.
9. A kit as claimed in claim 7 or 8, c h a r a c - 15 t e r i s e d in that the polyclonal activator is chosen amongst endotoxins, lectins, Staphylococcus aureus, pro¬ tein A or protein G.
10. A kit as claimed in claim 7, 8 or 9, c h a ¬ r a c t e r i s e d in that the polyclonal activator is
20 present in a suboptimal quantity.
11. A kit as claimed in one or several of claims 7, 9 and 10, c h a r a c t e r i s e d in that the lysosomo¬ tropic agents are lysosomotropic amino acid derivatives, or peptides based on such a derivative.
25 12. A kit as claimed in one or several of claims 7, 9 and 10, c h a r a c t e r i s e d in that the lysosomo¬ tropic agent is leucine-leucine-O-methyl ester, or pep¬ tides based on leucine-O-methyl ester.
30
35
PCT/SE1989/000486 1988-09-13 1989-09-12 In vitro immunisation of lymphocyte-containing cell populations and kit therefor WO1990002795A1 (en)

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SE8803208A SE461985B (en) 1988-09-13 1988-09-13 IN VITRO IMMUNIZATION OF Lymphocyte-containing CELL POPULATIONS AND MATERIAL KIT
SE8803208-1 1988-09-13

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EP0508472A3 (en) * 1991-04-10 1993-08-04 Kyowa Hakko Kogyo Co., Ltd. Method of producing hybridomas
WO2011020079A1 (en) 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
WO2012006596A2 (en) 2010-07-09 2012-01-12 Calmune Corporation Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
US11931383B2 (en) 2016-07-29 2024-03-19 Prokidney Bioactive renal cells for the treatment of chronic kidney disease

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AU645488B2 (en) * 1989-02-21 1994-01-20 Terumo Corporation A process for the generation of proliferating CD4 lymphocytes
CA2082041A1 (en) * 1990-05-22 1991-11-23 Paula Boerner Methods for producing antigen-specific, high-affinity human monoclonal antibodies

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0508472A3 (en) * 1991-04-10 1993-08-04 Kyowa Hakko Kogyo Co., Ltd. Method of producing hybridomas
WO2011020079A1 (en) 2009-08-13 2011-02-17 Calmune Corporation Antibodies against human respiratory syncytial virus (rsv) and methods of use
US9403900B2 (en) 2009-08-13 2016-08-02 Crucell Holland B.V. Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
US9988437B2 (en) 2009-08-13 2018-06-05 Janssen Vaccines & Prevention B.V. Anti-human Respiratory Syncytial Virus (RSV) antibodies and methods of use
WO2012006596A2 (en) 2010-07-09 2012-01-12 Calmune Corporation Anti-human respiratory syncytial virus (rsv) antibodies and methods of use
US9139642B2 (en) 2010-07-09 2015-09-22 Crucell Holland B.V. Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
US11931383B2 (en) 2016-07-29 2024-03-19 Prokidney Bioactive renal cells for the treatment of chronic kidney disease

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