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Specific human antibodies for selective cancer therapy

A selective and specific technology, applied in the field of peptides and polypeptides, can solve problems such as difficult separation, hindering repeated treatment, and reducing the therapeutic value of antibodies

Inactive Publication Date: 2004-12-01
BIO TECH GENERAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in-depth studies have shown that this method has several limitations
One limitation is the difficulty in isolating suitable monoclonal antibodies with selective binding capacity
The second limitation is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation
A third limitation is the induction of an immune response in patients to murine antibodies (human anti-mouse antibody-HAMA response), which often results in a short serum half-life and prevents repeated treatment, thus reducing the therapeutic value of said antibody
This HAMA response prevents repeated treatment and results in a short serum half-life of the product
Additionally, the methods described above can only isolate one type of antibody, and only against known and purified antigens
In addition, the method is not selective because the method can isolate antibodies against cell surface markers present on normal and malignant cells

Method used

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  • Specific human antibodies for selective cancer therapy
  • Specific human antibodies for selective cancer therapy
  • Specific human antibodies for selective cancer therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0336] 1. Preparation and protein purification of cells, bacterial strains, scFv phage display libraries, cell membranes for biopanning methods.

[0337] 1.1 Preparation of leukemia cells. Blood samples were obtained from leukemia patients. Monocytes (primary cells) were separated from other blood cells on Ficoll mats (Iso-prep, Robbins Scientific Corp., Sunnyvale, CA, USA). Centrifuge at 110 x g for 25 minutes. Cells at the interface were collected and washed twice with PBS. Cells were then suspended in RPMI+10% fetal calf serum (FCS) and counted. For long-term storage, 10% FCS and 10% DMSO were added to the lymphocytes and then frozen at -70°C.

[0338] 1.2 Preparation of fixed platelets. Platelet concentrates obtained from blood banks were incubated for 1 hour at 37°C. An equal volume of 2.0% paraformaldehyde was added and platelets were fixed at 40°C for 18 hours. Platelets were washed twice with cold saline (centrifuged at 2500 xg for 10 minutes). Resuspended in s...

Embodiment 2

[0344] 2. Manipulation of Phagemid Particles: Biopanning Method

[0345] 2.1 Phagemid screening and amplification: through a four-step biopanning method, the library is screened for phagemids expressing epitopes of particular interest:

[0346] a) binding phagemid particles to a target site, more specifically, binding said phagemid particles to washed target cells or cell membranes

[0347] b) Removal of unbound phagemid particles, more specifically, removal of unbound phagemid particles by extensive washing

[0348] c) Elution of bound phagemid particles

[0349] d) Propagation and amplification of eluted phagemid particles, more specifically, propagation and amplification in E. coli

[0350] 2.2 Cloning identification: The biopanning method of the four steps was basically repeated 3-5 times. Selected phagemid clones were propagated individually and further characterized by:

[0351] a) DNA sequencing

[0352] b) Ex vivo comparison of phage binding to several cell types ...

Embodiment 3

[0356] 3. Biopanning method

[0357] 3.1 Basic biopanning method: The biopanning method is an integral part of the above-mentioned phage display technology. Three biopanning methods were developed and employed in this study:

[0358] a) Method AM (AML membrane panning / bacterial elution followed by intact AML cell panning / trypsin elution)

[0359] b) Method YPR (Fixed Human Platelet Panning / Acid Elution)

[0360] c) Method YPNR (Fixed Human Platelet Panning / Acid Elution)

[0361] The above method is disclosed in detail below

[0362] 3.1.1 Method AM

[0363] 3.1.1.1 Pre-wash: Quickly thaw at 37°C containing 2×10 7 A 1 ml aliquot of frozen AML cells was diluted immediately into 10 ml of cold 2% PBS-milk (MPBS). Cells were centrifuged at 120 xg for 5 minutes at room temperature (RT), resuspended in MPBS, and counted with a hemocytometer. Prepare cell membranes as described in section 1.5.

[0364] 3.1.1.2 Selection was made by adding 2ml containing 10 12 Phagemid MPBS wa...

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Abstract

The present invention relates to a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of both, or a construct of a fragment, which has enhanced binding characteristics to favor selective and / or Or specifically bind to target cells, wherein the binding selectivity or specificity is mainly determined by the first hypervariable region, and wherein the Fv is scFv or dsFv, and optionally has one or more markers. Said enhanced binding is to a substantially exposed and / or overexpressed binding site on or within the target comprising a cell that is substantially Other cells that do not present and / or do not express the binding site are more favorable for binding. The invention also relates to methods for isolating said peptides and polypeptides from phage display libraries, and to nucleic acid molecules encoding them. The present invention provides pharmaceutical compositions comprising said peptides or polypeptides, and kits for the diagnosis and treatment of diseases, preferably cancer, most preferably acute myeloid leukemia.

Description

field of invention [0001] The present invention relates to the field of tissue targeting and identification by means of phage display technology, and to peptides and polypeptides capable of specifically binding to target cells. The peptides and polypeptides are Fv molecules, constructs thereof, fragments of both or constructs of fragments. More specifically, the peptides and polypeptides may have anticancer activity, and / or be associated or conjugated to anticancer agents, particularly against blood-related cancers. Background of the invention [0002] Tissue-selective targeting of therapeutic agents is an emerging discipline in the pharmaceutical industry. New guidance-based approaches to cancer therapy have been designed to increase the specificity and efficacy of therapy while reducing toxicity, thereby enhancing overall efficacy. To target toxins, radionucleotides, and chemotherapeutic conjugates to tumors, mouse monoclonal antibodies (MAb's) directed against tumor-ass...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K38/02A61K39/395A61K51/00A61P35/00C07K14/47C07K16/18C12N15/13C12P21/02C12P21/08C12Q1/02C12Q1/68
CPCA61K38/00C07K14/472A61P35/00C07K16/18
Inventor Y·哈盖J·拉扎罗维茨R·盖伊O·利普施茨E·桑顿A·莱瓦农D·普拉克辛T·佩雷茨
Owner BIO TECH GENERAL CORP
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