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Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders

A bispecific, combination technology, applied in the direction of drug combination, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of promoting allergic and inflammatory events

Active Publication Date: 2006-06-28
AMGEN RES (MUNICH) GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of high doses of protein or nucleic acid-based drugs include, inter alia, the promotion of allergic and inflammatory events, especially at the site of administration

Method used

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  • Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
  • Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
  • Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: CD19xCD3 and CD3xCD19 Single Chain Bispecifics Comprising Different Domain Rearrangements

[0083] Antibody construction

[0084] Typically, bispecific single chain antibody molecules comprising human CD3 antigen binding specificity domains and human CD19 antigen binding specificity domains are designed as shown in Table 1 below:

[0085] Constructed

No

SEQ ID Nos

(nuc / prot)

protein construct form

(N→C)

1

29 / 30

VL(CD19)-VH(CD19)-VH(CD3)-VL(CD3)

2

1 / 2

VH(CD19)-VL(CD19)-VH(CD3)-VL(CD3)

3

3 / 4

VL(CD19)-VH(CD19)-VL(CD3)-VH(CD3)

4

5 / 6

VH(CD19)-VL(CD19)-VL(CD3)-VH(CD3)

5

7 / 8

VL(CD3)-VH(CD3)-VH(CD19)-VL(CD19)

6

9 / 10

VH(CD3)-VL(CD3)-VH(CD19)-VL(CD19)

7

11 / 12

VL(CD3)-VH(CD3)-VL(CD19)-VH(CD19)

8

13 / 14

VH(CD3)-VL(CD3)-VL(CD19)-VH(CD19)

[0086]Variable light (VL) and variable heavy ...

Embodiment 2

[0097] Example 2: Expression and purification of single chain bispecific antibodies against CD3 and CD19

[0098] The protein was expressed in Chinese hamster egg cells (CHO). Expression vectors were transfected after treatment of cells with calcium phosphate ("Molecular Cloning", Sambrook et. al. 1989). Cells were cultured in CHO-modified DMEM medium in spinner flasks for 7 days prior to harvest. Cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20°C.

[0099] kta  FPLC system (Pharmacia) and Unicorn  Software is used for chromatography. All chemicals were of research grade and were purchased from Sigma (Deisenhofen) or Merck (Darmstadt). Load ZnCl with the manufacturer's protocol 2 Fractogel  The column (Merck) was subjected to solid phase metal affinity chromatography ("IMAC"). The column was equilibrated with A2 buffer (20 mM sodium phosphate buffer, pH 7.5, 0.4 M NaCl), and the cell culture supernatant (50...

Embodiment 3

[0107] Example 3: Flow cytometric binding analysis of CD19xCD3-specific polypeptides

[0108] To test the function of the constructs with respect to CD19 and CD3 binding capacity, flow cytometry analysis (FACS) was performed. For this purpose, CD19-positive Nalm 6 cells (human B-cell precursor leukemia) and CD3-positive Jurkat cells (human T-cell leukemia) were used. 200,000 Nalm 6 cells and 200,000 Jurkat cells were each incubated on ice for 30 min with 50 μl of pure cell supernatant from a culture of CHO cells expressing bispecifics with different arrangements of VH and VL domains for CD19 and CD3, respectively Antibodies (as described in Example 2). Cells were washed twice with PBS and binding of the constructs was detected as follows. Cells treated as described above were contacted with an unlabeled murine penta-histidine antibody (diluted 1:20 in 50 μl PBS with 2% FCS; Qiagen; Order No. 34660), which passed the C of the construct The terminal histidine tag specifically...

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Abstract

The present invention relates to a pharmaceutical composition comprising a bispecific single chain antibody construct comprising human CD3 and human CD19 specific binding domains, wherein the corresponding variable heavy chain regions (VH) and the corresponding variable light chain region (VL) from N-terminus to C-terminus in the following order: VH (CD19)-VL (CD19)-VH (CD3)-VL (CD3), VH (CD3)-VL (CD3)-VH (CD19)-VL (CD19) or VH (CD3)-VL (CD3)-VL (CD19)-VH (CD19). In addition, the present invention discloses the preparation method of the pharmaceutical composition and the medical / pharmaceutical use of the specific bispecific single chain antibody molecule specific to human CD3 antigen and human CD19 antigen.

Description

technical field [0001] The present invention relates to pharmaceutical compositions comprising bispecific single chain antibody constructs comprising binding domains specific for human CD3 and human CD19, wherein the corresponding variable heavy chain regions (V H ) and the corresponding variable light chain region (V L ) from N-terminal to C-terminal in the following order: V H (CD19)-V L (CD19)-V H (CD3)-V L (CD3), V H (CD3)-V L (CD3)-V H (CD19)-V L (CD19) or V H (CD3)-V L (CD3)-V L (CD19)-V H (CD19). In addition, the present invention discloses the preparation method of the pharmaceutical composition and the medical / pharmaceutical use of the bispecific single-chain antibody molecule specific for human CD3 antigen and human CD19 antigen. Background technique [0002] Despite their medical importance, research on B cell-mediated diseases such as non-Hodgkin lymphoma has yielded only a small amount of clinically available data, and conventional approaches to tre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P19/02A61P35/00A61P35/02
CPCA61K2039/505C07K16/2803C07K2317/31C07K2317/622C07K2317/34A61P19/02A61P29/00A61P31/00A61P31/04A61P31/12A61P33/00A61P35/00A61P35/02A61P37/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00
Inventor P·库弗R·卢特比泽B·科赫莱森S·泽曼P·博伊尔勒
Owner AMGEN RES (MUNICH) GMBH
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