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Recombinant proteins and their therapeutic uses

A recombinant protein and protein technology, applied in the field of recombinant protein for the treatment of diseases, can solve problems such as no clinical effect

Inactive Publication Date: 2014-09-24
BIOVEN 3 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the target polypeptide and carrier protein molecules can be coupled to themselves, resulting in multiple homomultimers that may not have clinical effects and may not generate an anticancer immune response in the patient

Method used

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  • Recombinant proteins and their therapeutic uses
  • Recombinant proteins and their therapeutic uses
  • Recombinant proteins and their therapeutic uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0099] Example I: ELISA protocol

[0100] To determine whether a recombinant protein, such as the synthetic EGF-CT-B protein according to the present disclosure, is capable of displaying the B-loop of EGF in the correct conformation, two commercially available monoclonal antibodies (Santa Cruz Antibodies) were obtained , Cat. Nos. 10825 and 10827), two monoclonal antibodies known to block the binding of EGF to the EGF receptor. Without being bound by any particular theory, it is hypothesized that a number of sources of binding to the EGF receptor are in part via the region defined by residues Met21-Ala30.

[0101] In an exemplary embodiment, mAb 10825 and mAb 10827 at concentrations of 1 ug / ml and 2 ug / ml were used to bind recombinant EGF (rEGF) protein in an ELISA and optical density (OD) was measured at 450 nm. refer to Figure 6 The results are illustrated in a histogram. Such as Figure 6 It was shown that when rEGF was adsorbed on the ELISA plate, rEGF retained its na...

Embodiment II

[0105] Example II: Presentation of EGF Neutralizing Epitopes

[0106]To determine whether the recombinant protein EGF-CT-B vaccine expressing EGF at the end of the CT-B sequence interferes or affects any desired intrinsic properties of the EGF domain, in particular the presentation of the correct conformation of the Met21-Ala30 epitope of EGF , and whether it interferes or affects the ability of CT-B monomers to assemble into multimers (pentameric rings) under suitable physical-chemical conditions, and created 6 recombinant proteins, which are in CT-B The N-terminus (Test 1 to Test 3) or C-terminus (Test 4 to Test 6) of the sequence expressed the entire EGF coding region.

[0107] Test 1 and Test 4 contained the recombinant protein EGF-CT-B vaccine expressing the full-length EGF sequence directly on the CT-B domain. Test 2 and Test 5 contained a synthetic EGF-CT-B vaccine expressing the full-length EGF sequence separated from the CT-B domain by a short 3 amino acid peptide se...

Embodiment III

[0115] Example III: Multimer assembly of EGF-CT-B protein

[0116] To examine the effect of expressing domains including growth factors at the termini of CT-B-derived recombinant proteins on monomer-subunit assembly into multimers, synthetic proteins were made to test 1 under native conditions (non-reducing, not boiled). Run to test 6 on SDS-PAGE gel. Synthetic recombinant EGF-CT-B protein was then transferred to nitrocellulose membranes by electroblotting and probed with rabbit anti-CT-B antibody (as described in Example II). Binding of the HRP-labeled secondary anti-rabbit antibody was detected via light emission from the ECL substrate on the imaging membrane. Such as Figure 10 As shown, Western blotting confirmed the presence of high molecular weight CT-B, suggesting that the synthesized EGF-CT-B monomeric protein was able to assemble into multimers via the CT-B domain.

[0117] In separate experiments, replicate samples of native (non-boiled or non-reduced) CT-B protei...

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Abstract

A recombinant protein expressing one or more human growth factors, tumor antigens, and / or receptors or epitopes thereof on or within an immunogenic expression creating a recombinant protein in which one or more epitopes are presented on the surface of the sequence in their natural configuration. The growth factor, tumor antigen, and / or receptor, sequence(s) may be expressed within the encoding sequence at appropriate internal positions or at the termini as single expressions or as two or more tandem repeats.

Description

technical field [0001] The present invention relates to the field of recombinant protein for treating diseases. [0002] Cross References to Related Applications [0003] This application claims priority to U.S. Patent Application Serial No. 61 / 563,128, filed November 23, 2011, entitled "Synthetic Recombinant Proteins for Immunogenicity," and filed June 1, 2012, entitled "Immunogenic Original Synthetic Recombinant Proteins," US Patent Application Serial No. 61 / 654,401, both of which are hereby incorporated by reference in their entirety. Background technique [0004] Cancer immunology is the study of the interaction between the immune system and cancer cells (eg, tumors or malignancies). The initiation of an immune response, such as the recognition of cancer-specific antigens, which are expressed by human tumors and not expressed in normal tissues, is of particular importance. Typically, the approach to control malignant cell division and proliferation is to isolate and p...

Claims

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Application Information

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IPC IPC(8): A61K39/385C07K14/435
CPCC07K14/48A61K2039/70A61K39/0011C07K14/475A61K2039/645C07K14/4748C07K14/485C07K14/71C07K14/50C07K14/28C07K14/4753A61K39/0005C12N9/12C12N9/6424A61P31/04A61P35/00A61P37/04A61P43/00Y02P20/582C07K14/495C07K14/65C07K2319/40
Inventor 基思·艾伦·查尔顿E·德杭特
Owner BIOVEN 3
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