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Methods of treating epidermal growth factor deletion mutant VIII related disorders

A technology of antibody and body weight, applied in chemical instruments and methods, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, etc., can solve the problem of low antibody affinity

Inactive Publication Date: 2014-11-26
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, even after affinity maturation, the affinity of such antibodies is relatively low, approximately 2.2 × 10 -8 to 1.5×10 -9

Method used

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  • Methods of treating epidermal growth factor deletion mutant VIII related disorders
  • Methods of treating epidermal growth factor deletion mutant VIII related disorders
  • Methods of treating epidermal growth factor deletion mutant VIII related disorders

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0234] Antigen preparation

[0235] a. Preparation of EGFRvIII PEP3-KLH antigen

[0236] Related to Example 2, a 14-mer human EGFRvIII PEP3 (LE E K K G N Y V V T D H C (SEQ ID NO: 56)) peptide was custom-synthesized by the R&D system. The PEP3 peptide was then conjugated to keyhole limpet hemocyanin (KLH) as follows: EGFRvIII PEP3 (200mcg) (R&D) was mixed with 50mcg keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL) to a final concentration of 165mcl using distilled water. volume. Add 250mcl conjugation buffer (0.1M MES, 0.9M NaCl, pH4.7) and add 10mg / ml stock solution (EDC, Pierce, Rockford, IL) cross-linked EGFRvIII PEP3 and KLH. The conjugate was incubated at room temperature for 2 hours and unreacted EDC was removed by centrifugation through a 1 kDa filter (centrifugal filter; Millipore, Bedford, MA) using PBS pH 7.4.

[0237] Related to Example 3, a 14-mer human EGFRvIII PEP3 (LE E K K G N Y V V T D H C (SEQ ID NO: 56)) peptide was custom synthesized. The PEP3 ...

Embodiment 2

[0280] Generation of anti-EGFRvIII antibodies by generation of hybridomas

[0281] For this approach, eight XenoMouse mice producing antibodies with the gamma-1 constant region (XenoMouse G1 mice) were immunized on day 0 and boosted on days 11, 21, 32, 44 and 54 and on day 58. days for fusion. All immunizations were performed by subcutaneous administration at the base of the tail plus intraperitoneal administration for all injections. Suspended in 1:1 v / v mixed with complete Freunds adjuvant (complete Freunds adjuvant) (CFA) (Sigma, St.Louis, MO) in pyrogen-free DPBS 1.5 × 10 7 B300.19 / EGFRvIII transfected cells (Example 1A) were immunized on day 0. 1.5×10 in DPBS mixed with incomplete Freunds adjuvant (IFA) (Sigma, St.Louis, MO) 1:1 v / v 7 B300.19 / EGFRvIII transfected cells were boosted on days 11, 21 and 32. A boost on day 44 was performed with 5 μg of PEP3 (EGFRvIII peptide)-KLH conjugate (Example 1) in DPBS mixed with IFA at 1:1 v / v, and 5 μg of PEP3 in DPBS without a...

Embodiment 3

[0290] Generate antibodies by using XenoMax technology

[0291] Immunization of XenoMouse Animals

[0292] By sequentially making XenoMouse mice producing antibodies with a γ-1 constant region (XenoMouse G1 mice), XenoMouse mice producing antibodies with a γ-2 constant region (XenoMouse XMG2 mice), and producing antibodies with a γ-4 constant region XenoMouse mice (XenoMouse G4 mice) were immunized with antibodies to develop human monoclonal antibodies against human EGFRvIII.

[0293] For generating mAbs by XenoMax technology, EGFRvIII PEP3 (Example 1A) and 300.19 cells expressing EGFRvIII (Example 1B) or the extracellular domain (EGFRvIII-ECD) of the bacterially expressed EGFRvIII protein (Dr. Bigner, Duke University, USA) were used. ) and 300.19 cells expressing EGFRvIII or with EGFRvIII-rabbit Fc fusion protein (EGFRvIII-RbFc) (Example 1C) and 300.19 cells expressing EGFRvIII or with EGFRvIII-RbFc, via paw only (FP) or via base of tail by subcutaneous injection and intr...

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Abstract

The present invention relates to methods of treating treating epidermal growth factor deletion mutant vIII (EGFRvIII) related disorders, such as glioblastoma or anaplastic astrocyte tumors, using antigen binding proteins, including antibodies against EGFRvIII conjugated to a drug. Diagnostic and therapeutic formulations of such antibodies and drug conjugates thereof are also provided.

Description

[0001] This application claims the benefit of US Provisional Application No. 61 / 727,029, filed November 15, 2012, and US Provisional Application No. 61 / 560,731, filed November 16, 2011, which are incorporated herein by reference. [0002] Reference to Sequence Listing [0003] This application is filed in electronic format together with a Sequence Listing. The sequence listing is provided as a file named A-1680-US-PSP_SEQ.txt created on November 16, 2011, 89 KB in size. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety. technical field [0004] The present invention relates to methods of treating epidermal growth factor deletion mutant vIII (EGFRvIII)-associated diseases, such as glioblastoma or anaplastic astrocytoma, using antigen-binding proteins, including drug-conjugated anti-EGFRvIII antibodies. Diagnostic and therapeutic formulations of such antibodies and drug conjugates are thus also provided. Backgrou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61P35/00C07K16/28C07K16/30
CPCC07K16/30C07K2317/34C07K2317/73C07K2317/21C07K2317/77A61K45/06A61K2039/505A61K47/48446C07K16/2863C07K2317/33A61K47/48561A61K47/6819A61K47/6849A61P35/00A61K47/50A61K39/395C07K16/28
Inventor J.S.希尔K.J.汉布莱特
Owner AMGEN INC
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