Method for ex vivo immunization using heterologous intact bispecific and/or trispecific antibodies
View Patent ↗According to the invention there is described a method for ex vivo immunization of humans and animals comprising the following steps: a) isolating autologous tumor cells; b) treating the tumor cells to prevent the survival thereof following reinfusion; c) incubating the thus treated tumor cells with intact heterologous bispecific and/or trispecific antibodies showing the following properties: α—binding to a T cell; β—binding to at least one antigen on a tumor cell; γ—binding, by their Fc portion (in the case of bispecific antibodies), or by a third specificity (in the case of trispecific antibodies) to Fc receptor-positive cells.
1. A method for the preparation of an antibody-tumor cell preparation for immunization of humans and animals against tumor cells comprising the steps of:
a) isolating autologous tumor cells;
b) treating the tumor cells to prevent the survival thereof following reinfusion;
c) incubating the thus treated tumor cells with intact heterologous bispecific antibodies showing the following properties:
(i) binding to a T cell;
(ii) binding to at least one tumor-associated antigen on a tumor cell;
(iii) binding, by their Fc portion to Fc receptor-positive cells; and
(iv) capable of activating the Fc receptor-positive cell whereby the expression of cytokines, co-stimulatory antigens or both is induced or increased,
wherein the bispecific antibodies have isotype combinations selected from the group consisting of:
rat-IgG2b/human-IgG1,
rat-IgG2b/human-IgG2,
rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],
rat-IgG2b/human-IgG4,
rat-IgG2b/rat-IgG2c,
mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding to protein A, in the following indicated as *],
mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, oriental allotype],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3],
human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2 [N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
rat-IgG2b/mouse-IgG2a,
rat-IgG2b/mouse-IgG2b, and
rat-IgG2b/mouse-IgG3.
2. The method according to claim 1 , in which said antibodies bind Fc receptor-positive cells having a Fcγ receptor I, II, or III.
3. The method according to claim 2 , in which said Fcγ receptor I-positive cells are selected from the group consisting of monocytes, macrophages, dendritic cells, and activated neutrophils.
4. The method according to claim 1 , in which said antibodies induce tumor-reactive complement-binding antibodies, thereby inducing a humoral immune response.
5. The method according to claim 1 , in which said antibodies bind to the T cells via CD2, CD3, CD4, CD5, CD6, CD8, CD28 or CD44.
6. The method according to claim 1 , in which said antibodies, following their binding to the Fc receptor-positive cells, initiate or increase the expression of co-stimulatory antigens CD40, CD80, CD86, ICAM-1 and/or LFA-3 and/or initiate or increase the secretion of cytokines by the Fc receptor-positive cells.
7. The method according to claim 1 , in which said antibodies increase the secretion of IL-1, IL-2, IL-4, IL-6, IL-8, IL-12 or TNF-α or a combination thereof.
8. The method according to claim 1 , in which said bispecific antibody is an anti-CD3 X anti-tumor-associated antigen antibody or anti-CD4 X anti-tumor-associated antigen antibody or anti-CD5 X anti-tumor-associated antigen antibody or anti-CD6 X anti-tumor-associated antigen antibody or anti-CD8 X anti-tumor-associated antigen antibody or anti-CD2 X anti-tumor-associated antigen antibody or anti-CD28 X anti-tumor-associated antigen antibody or anti-CD44 X anti-tumor-associated antigen antibody.
9. The method according to claim 1 , in which said tumor cells are incubated with the antibodies for a period of 10 minutes to 5 hours.
10. The method according to claim 1 , in which said tumor cells are incubated with the antibodies for a period of 15 minutes to 120 minutes.
11. The method according to claim 1 , in which said tumor cells are present in the amount of about 10 7 to 10 9 cells.
12. The method according to claim 1 , in which said bispecific antibodies are added in an amount of 2 to 100 μg.
13. The method according to claim 1 , in which said treating of the tumor cells in step b is performed by irradiation.
14. A method for preparing a vaccine comprising an antibody-tumor cell preparation, said method comprising preparing an antibody-tumor cell preparation by the method of claim 1 , and preparing a vaccine from said antibody-tumor cell preparation.
15. A method for preparing a vaccine comprising activated peripheral blood mononucleated cells, said method comprising preparing an antibody-tumor cell preparation by a method comprising steps (a) and (b) of the method of claim 1 and further comprising the step of incubating the thus-treated tumor cells with both intact heterologous bispecific antibodies and peripheral blood mononucleated cells, thereby activating said peripheral blood mononucleated cells, and preparing a vaccine from the thus-activated peripheral blood mononucleated cells, wherein said intact heterologous bispecific antibodies have the following properties:
(i) binding to a T cell;
(ii) binding to at least one tumor-associated antigen on a tumor cell;
(iii) binding by their Fc portion to Fc receptor-positive cells; and
(iv) capable of activating the Fc receptor-positive cell whereby the expression of cytokines, co-stimulatory antigens or both is induced or increased,
and said bispecific antibodies have isotype combinations selected from the group consisting of:
rat-IgG2b/human-IgG1,
rat-IgG2b/human-IgG2,
rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],
rat-IgG2b/human-IgG4,
rat-IgG2b/rat-IgG2c,
mouse-IgG2a/human-IgG3 [caucasian allotypes G3m(b+g)=no binding to protein A, in the following indicated as *],
mouse-IgG2a/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2a/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, oriental allotype],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3],
human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG2/human-[VH-CH1, VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
mouse-IgG2b/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/human-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
rat-IgG2b/mouse-IgG2a,
rat-IgG2b/mouse-IgG2b, and
rat-IgG2b/mouse-IgG3.
16. The method according to claim 15 in which said incubation of step (d) is performed for a period of 1 to 14 days.
17. The method according to claim 15 in which said incubation of step (d) is performed with about 10 8 to 10 10 mononucleated peripheral cells.
18. The method of claim 15 , wherein the peripheral blood mononucleated cells are added following a preincubation of the thus-treated tumor cells with said intact heterologous bispecific antibodies.
19. A method for preventing the reoccurrence of a tumor, said method comprising administering an antibody-tumor cell preparation prepared according to the method of claim 1 to an individual in whom tumor cells have reappeared.
20. A pharmaceutical composition comprising an antibody-tumor cell preparation obtained by the method of claim 1 .
21. A method for preventing the recurrence of a tumor, said method comprising: preparing an antibody-tumor cell preparation by the method of claim 1 in which step (c) is replaced with step (d), which comprises incubating the thus-treated tumor cells with both intact heterologous bispecific antibodies and peripheral blood mononucleated cells, thereby activating said peripheral blood mononucleated cells; and administering to an individual in whom tumor cells have reappeared the activated peripheral blood mononucleated cells, wherein said intact heterologous bispecific antibodies have the following properties:
(i) binding to a T cell;
(ii) binding to at least one tumor-associated antigen on a tumor cell;
(iii) binding, by their Fc portion to Fc receptor-positive cells; and
(iv) capable of activating the Fc receptor-positive cell whereby the expression of cytokines, co-stimulatory antigens or both is induced or increased,
and said bispecific antibodies have isotype combinations selected from the group consisting of:
rat-IgG2b/human-IgG1,
rat-IgG2b/human-IgG2,
rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],
rat-IgG2b/human-IgG4,
rat-IgG2b/rat-IgG2c,
mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding to protein A, in the following indicated as *],
mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, oriental allotype],
rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3],
human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4 [N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3]
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: >aa position 251]-human-IgG3*[CH3],
mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],
mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],
rat-IgG2b/mouse-IgG2a,
rat-IgG2b/mouse-IgG2b, and
rat-IgG2b/mouse-IgG3.
22. The method of claim 21 , wherein the peripheral blood mononucleated cells are added following a preincubation of the thus-treated tumor cells with said intact heterologous bispecific antibodies.