Method for ex vivo immunization using heterologous intact bispecific and/or trispecific antibodies

• US 7,018,632 B2
• Filed: 06/15/1998
• Issued: 03/28/2006
• Est. Priority Date: 06/17/1997
• Status: Expired due to Fees

• Current Assignee: Helmholtz Zentrum München Deutsches Forschungszentrum für. Gesundheit und Umwelt (GmbH)
• Original Assignee: Gsf-Forschungszentrum Fur Umwelt Und Gesundheit GmbH
• Inventors: Zeidler, Reinhard, Bornkamm, Georg, Kolb, Hans-Joachim, Lindhofer, Horst
• Primary Examiner(s): Harris, Alana M.
• Assistant Examiner(s): Holleran, Anne L.
• Application Number:
  US09/094,921
• Publication Number:
  US 20020009430A1
• Time in Patent Office: 2,843 Days
• Field of Search: 424/277.1, 424/130.1, 424/133.1, 424/136.1, 424/138.1, 424/172.1, 424/173.1, 424/174.1, 424/93.71, 424/132.1, 424/135.1, 424/141.1, 424/143.1, 424/144.1, 424/152.1, 424/153.1, 424/154.1, 424/155.1, 424/156.1, 424/93.1, 424/93.21, 424/93.3, 424/93.7, 530/387.3
• US Class Current: 424/136.1
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/13 Tumour cells, irrespective of tissue of origin tumour vaccines A61K39/00
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 41/17 by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

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.

Claims

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 10⁷ to 10⁹ cells.

12. The method according to claim 1, in which said bispecific antibodies are added in an amount of 2 to 100 μ

g.

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 10⁸ to 10¹⁰ 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.