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Небесная энциклопедия

Космические корабли и станции, автоматические КА и методы их проектирования, бортовые комплексы управления, системы и средства жизнеобеспечения, особенности технологии производства ракетно-космических систем

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Мониторинг СМИ

Мониторинг СМИ и социальных сетей. Сканирование интернета, новостных сайтов, специализированных контентных площадок на базе мессенджеров. Гибкие настройки фильтров и первоначальных источников.

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Форма поиска

Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка
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Применить Всего найдено 8933. Отображено 100.
19-04-2012 дата публикации

Mice That Make VL Binding Proteins

Номер: US20120096572A1
Автор: Andrew J. Murphy, Cagan Gurer, Karolina A. Hosiawa, Lynn Macdonald, Sean Stevens
Принадлежит: Regeneron Pharmaceuticals Inc

Genetically modified mice and methods for making an using them are provided, wherein the mice comprise a replacement of all or substantially all immunoglobulin heavy chain V gene segments, D gene segments, and J gene segments with at least one light chain V gene segment and at least one light chain J gene segment. Mice that make binding proteins that comprise a light chain variable domain operably linked to a heavy chain constant region are provided. Binding proteins that contain an immunoglobulin light chain variable domain, including a somatically hypermutated light chain variable domain, fused with a heavy chain constant region, are provided. Modified cells, embryos, and mice that encode sequences for making the binding proteins are provided.

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Номер записи: 1
14-06-2012 дата публикации

method of producing an antibody using a cancer cell

Номер: US20120151611A1
Автор: Hiroyuki Satofuka, Masahiro Uchino, Shingo Hanaoka
Принадлежит: Individual

The present invention aims to provide a method for antibody preparation. The present invention is directed to a method for preparing an antibody-producing cell, which comprises the following steps: (1) transplanting metastatic cancer cells capable of expressing a target antigen into a non-human animal to ensure engraftment of the cancer cells in the animal; (2) immunizing the animal with the target antigen; and (3) collecting the antibody-producing cell from the immunized animal; as well as a method for preparing an antibody, which comprises collecting the antibody from the antibody-producing cell prepared by the above method.

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Номер записи: 2
19-07-2012 дата публикации

Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation

Номер: US20120183997A1
Автор: Brian Toki, Dennis R. Benjamin, Django Sussman, Patrick J. Burke, Scott C. Jeffrey, Stephen C. Alley
Принадлежит: Seattle Genetics Inc

The invention provides methods and compositions for preparing antibodies and antibody derivatives with reduced core fucosylation.

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Номер записи: 3
06-09-2012 дата публикации

Stable formulations of immunoglobulin single variable domains and uses thereof

Номер: US20120225072A1
Автор: Ann Brige, Yves Meyvis
Принадлежит: Ablynx NV

The present invention relates to stable formulations of polypeptides, e.g. immunoglobulin single variable domains.

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Номер записи: 4
13-09-2012 дата публикации

CH2 Domain Template Molecules Derived From Rational Grafting Of Donor Loops Onto CH2 Scaffolds

Номер: US20120230981A1
Автор: David Bramhill, Gopalan Raghunathan
Принадлежит: Research Corp Technologies Inc

Novel CH2 domain template molecules wherein donor loops from a database of domains are transferred to a CH2 domain scaffold. At least one or up to three loops from a donor are transferred to the CH2 domain. The donor loops may be chosen based on length, e.g., the donor loop may have a length that is similar to that of a structural loop in the CH2 domain scaffold.

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Номер записи: 5
22-11-2012 дата публикации

N-acetylhexosamine-containing n-glycans in glycoprotein products

Номер: US20120295273A1
Автор: Brian E. Collins, Carlos J. Bosques, Enrique Arevalo, Jay Duffner, Kevin Millea, Nathaniel J. Washburn
Принадлежит: Momenta Pharmaceuticals Inc

The present invention provides methods of evaluating a glycoprotein preparation for the absence, presence or amount of an N-acetylhexosamine glycan, e.g., an N-acetylglucosamine glycan.

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Номер записи: 6
29-11-2012 дата публикации

Focused libraries of genetic packages

Номер: US20120302463A1
Автор: Robert Charles Ladner
Принадлежит: Dyax Corp

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies.

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Номер записи: 7
27-12-2012 дата публикации

Novel Lowered Affinity Antibodies And Methods of Making the Same

Номер: US20120329995A1
Автор: Hsiu-Ching Chang
Принадлежит: AB Biosciences Inc

The present invention provides methods for making novel, rationally designed lowered affinity antibodies. The methods of the present invention make antibodies that have variable domains that have been designed to reduce or eliminate the antigen binding activity of the parental antibody without altering the overall (3) dimensional antibody structure. Using the antibodies made using methods of the present invention in various assays allows researchers to distinguish effects that result from specific antigen-antibody interactions from other, non-specific antibody effects.

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Номер записи: 8
17-01-2013 дата публикации

Method for the production of proteins and peptides

Номер: US20130017576A1
Автор: Christine Gless, Helmut Merk, Michael Gerrits, Wolfgang Stiege
Принадлежит: Rina Netzwerk Rna-Technologien Gmbh

The invention relates to a method for producing monomeric or dimeric proteins or peptides containing internal or external disulfide bonds, comprising the following steps: a) a cell-free lysate, obtainable from eukaryotic cells, is provided, which contains functional microsomal vesicles, b) a nucleic acid coding the protein or peptide and additionally containing a signal sequence is added to the lysate, c) the lysate with the nucleic acid is held for a given time at a temperature in the range from 20 to 35° C., proteins or peptides formed with the nucleic acid being translocated into the microsomal vesicles, d) the microsomal vesicles are then dissolved, and the proteins or peptides obtained thereby are optionally separated from the lysate.

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Номер записи: 9
21-03-2013 дата публикации

METHOD FOR PREPARING ANTIBODIES HAVING IMPROVED PROPERTIES

Номер: US20130071390A1
Автор: Liu Liming, Stadheim Terrance A., Zha Dongxing
Принадлежит: MERCK, SHARP & DOHME CORP

The present invention is directed to methods and compositions for the production of Fc-containing polypeptides having improved properties and comprising mutations at positions 243 and 264 of the Fc region. 1. An Fc-containing polypeptide comprising mutations at amino acid positions 243 and 264 of the Fc region , wherein the Fc-containing polypeptide is an antibody or antibody fragment comprising sialylated N-glycans , wherein the mutations are F243A and V264A , and wherein the numbering is according to the EU index as in Kabat.2. (canceled)3. (canceled)4. (canceled)5. The Fc-containing polypeptide of claim 1 , wherein the sialic acid residues in the sialylated N-glycans are attached via α-2 claim 1 ,6 linkages.6. The Fc-containing polypeptide of or claim 1 , wherein the Fc-containing polypeptide has one or more of the following properties when compared to a parent Fc-containing polypeptide:a) reduced effector function,b) increased anti-inflammatory properties,c) increased sialylation,d) increased bioavailability when administered parenterally, ande) reduced binding to FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa.7. A method for producing a Fc-containing polypeptide in a host cell comprising:a) providing a genetically modified cell that has been genetically engineered to produce an Fc-containing polypeptide, wherein the Fc-containing polypeptide is an antibody or an antibody fragment comprising sialylated N-glycans, wherein the host cell comprises a nucleic acid encoding mutations at amino acid positions 243 and 264 of the Fc region of the Fc-containing polypeptide, wherein the mutations are F243A and V264A, and wherein the numbering is according to the EU index as in Kabat;b) culturing the host cell under conditions which cause expression of the Fc-containing polypeptide; andc) isolating the Fc-containing polypeptide from the host cell.8. (canceled)9. (canceled)10. (canceled)11. The method of claim 7 , wherein the sialic acid residues in the sialylated N-glycans are ...

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Номер записи: 10
04-04-2013 дата публикации

COMPOSITIONS AND METHODS FOR INCREASING SERUM HALF-LIFE

Номер: US20130084291A1
Автор: Knopf John, Kumar Ravindra, Seehra Jasbir, Vogan Erik
Принадлежит: Acceleron Pharma, Inc.

Provided herein are glycovariant Fc fusion proteins having increased serum half lives. Also provided are methods for increasing the serum half life of an Fc fusion protein by introducing one or more non-endogenous glycosylation sites. 1. A polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 47.2. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:47.3. The polypeptide of claim 1 , wherein the polypeptide further comprises a constant domain of an immunoglobulin heavy chain.4. The polypeptide of claim 3 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:48.5. A polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:48.6. A fusion protein comprising an immunoglobulin Fc domain and at least one heterologous polypeptide domain claim 3 , wherein the heterologous polypeptide domain has an amino acid sequence that is at least 90% claim 3 , 95% claim 3 , 96% claim 3 , 97% claim 3 , 98% or 99% identical to SEQ ID NO:38 claim 3 , wherein the fusion protein is modified outside of the immunoglobulin Fc domain to introduce at least one non-endogenous N-linked glycosylation site claim 3 , and wherein glycosylation at the one or more introduced glycosylation sites increases the serum half-life of the modified fusion protein by at least 10% relative to the serum half-life of the fusion protein lacking an introduced glycosylation site as measured in a pharmacokinetic monkey assay.7. The fusion protein of claim 6 , wherein the glycosylation site is introduced in the extracellular domain outside of the ligand binding pocket.8. The fusion protein of claim 6 , wherein glycosylation at the one or more introduced glycosylation sites does not affect ligand binding activity of the receptor by more than 3-fold.9. The fusion protein of claim 6 , wherein the fusion protein is modified by addition or ...

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Номер записи: 11
11-04-2013 дата публикации

Hybrid Constant Regions

Номер: US20130089547A1
Автор: Tso J. Yun, Tsurushita Naoya
Принадлежит: JN Biosciences LLC

The invention provides hybrid constant regions and antibodies or fusion proteins incorporating the same. The hybrid constant regions include at least CH2 and CH3 regions of an IgG or IgA constant region and Cμ3 and Cμ4 regions of a Cμ constant region. The hybrids retain properties of both component constant regions. The hybrids retain the ability of a Cμ constant region to form multivalent complexes, e.g., pentameric or hexameric structures. IgG hybrids also retain IgG properties including pH-dependent FcRn binding, which is associated with a relatively long in vivo half-life, and specific binding to protein G, which facilitates purification. Depending on the isotype and subtype, the nature of the antigen and presence of additional IgG CH1 and hinge domains, IgG hybrids may also retain properties of specific binding to protein A, and effector functions ADCC, CDC and opsonization. IgA hybrids retain the property of IgA of binding to an Fc-alpha receptor CD89. 1. An antibody or fusion protein comprising an immunoglobulin heavy chain constant region , comprising in order from N- to C-terminus CH2 and CH3 regions , each of which is of IgG or IgA isotype , and Cμ3 and Cμ4 regions.2. The antibody or fusion protein of claim 1 , wherein the immunoglobulin heavy chain further comprises a hinge region N-terminal to the CH2 region.3. The antibody or fusion protein of claim 1 , wherein the immunoglobulin heavy chain further comprises a CH1 region N-terminal to the hinge region.4. The antibody or fusion protein of claim 3 , which is an antibody claim 3 , wherein the heavy chain constant region is fused to a heavy chain variable region and the antibody further comprises a light chain comprising a light chain variable region and constant region.5. The antibody of as a component of a multi-specific antibody comprising a plurality of such antibodies with different heavy chain variable regions claim 4 , and optionally different light chain variable regions; the plurality of ...

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Номер записи: 12
18-04-2013 дата публикации

METHODS AND COMPOSITION FOR SECRETION OF HETEROLOGOUS POLYPEPTIDES

Номер: US20130096280A1
Автор: Marrichi Matthew, REILLY Dorothea E.
Принадлежит: Genentech, Inc.

The present invention relates generally to the fields of molecular biology and protein technology. More specifically, the invention concerns signal sequences for the secretion of heterologous polypeptide from bacteria. The invention also concerns recombinant polypeptides and uses thereof. 1. A method of making an a heterologous polypeptide , said method comprising culturing a host cell comprising polynucleotide comprising a variant translation initiation region (TIR) comprising nucleic acid variants of a PhoA , MalE , DsbA or STII secretion signal region operably linked to a polynucleotide encoding the heterologous polypeptide , so that the polynucleotide is expressed , whereby upon expression of the heterologous protein in a host cell , the heterologous polypeptide is folded to form a biologically active heterologous polypeptide.2. The method of claim 1 , wherein the variant TIR comprises a sequence of one of SEQ ID NOs 1-42.3. The method of claim 1 , wherein the variant TIR comprises sequence of one of SEQ ID NOs. 1-14 claim 1 , 16-24 claim 1 , 26-39 claim 1 , 41-42.4. The method of claim 1 , wherein the translational strength of said variant TIR 15 less than the translational strength of the wild-type TIR.5. The method of claim 1 , wherein the translational strength of said variant TIR 15 greater than the translational strength of the wild-type TIR.6. A method of making an antibody claim 1 , said method comprising culturing a host cell comprising a polynucleotide comprising (1) a first TIR operably linked to a polynucleotide encoding an antibody heavy chain claim 1 , wherein the TIR comprises a co-translational prokaryotic secretion signal sequence; and (2) a second TIR operably linked to a polynucleotide encoding an antibody light chain claim 1 , wherein the second TIR comprises a co-translational or post-translational prokaryotic secretion signal sequence claim 1 , so that the antibody is expressed claim 1 , whereby upon expression of the antibody in the host ...

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Номер записи: 13
25-04-2013 дата публикации

ANTIBODY CONSTANT REGION VARIANT

Номер: US20130101581A1
Автор: Igawa Tomoyuki, Kuramochi Taichi, Maeda Atsuhiko, Mimoto Futa
Принадлежит: Chugai Seiyaku Kabushiki Kaisha

The present inventors carried out dedicated research to generate antibody constant regions with reduced Fcγ receptor-binding activity by altering amino acid sequences in the antibody constant region. As a result, the present inventors successfully identified novel constant region sequences with reduced Fcγ receptor-binding activity compared to conventional antibody constant regions. 1. A polypeptide comprising an antibody constant region of any one of (a) to (m) below:(a) an antibody constant region comprising an amino acid sequence comprising substitutions of Leu at position 234 (EU numbering), Leu at position 235 (EU numbering), and Asn at position 297 (EU numbering) in the amino acid sequence of SEQ ID NO: 5 (IgG1 constant region) with other amino acids;(b) an antibody constant region comprising an amino acid sequence comprising substitutions of Leu at position 234 (EU numbering), Leu at position 235 (EU numbering), Ala at position 327 (EU numbering), Ala at position 330 (EU numbering), and Pro at position 331 (EU numbering) in the amino acid sequence of SEQ ID NO: 5 (IgG1 constant region) with other amino acids;(c) an antibody constant region comprising an amino acid sequence comprising substitutions of Leu at position 234 (EU numbering), Leu at position 235 (EU numbering), Asn at position 297 (EU numbering), Ala at position 327 (EU numbering), Ala at position 330 (EU numbering), and Pro at position 331 (EU numbering) in the amino acid sequence of SEQ ID NO: 5 (IgG1 constant region) with other amino acids;(d) an antibody constant region comprising an amino acid sequence comprising substitutions of Leu at position 234 (EU numbering), Leu at position 235 (EU numbering), Ala at position 327 (EU numbering), Ala at position 330 (EU numbering), Pro at position 331 (EU numbering), and Asn at position 434 (EU numbering) in the amino acid sequence of SEQ ID NO: 5 (IgG1 constant region) with other amino acids;(e) an antibody constant region comprising an amino acid ...

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Номер записи: 14
25-04-2013 дата публикации

SELF-ASSEMBLING HALF-ANTIBODIES

Номер: US20130101665A1
Автор: Chevillard Sylvie, Falck Caroline, Lefevre Emilie, Tisne-Vicrobeck Carine, Ugolin Nicolas
Принадлежит: Centre National De La Recherche Scientifique

Novel chimeric molecules, termed “half-antibodies”, which are capable of self-assembling to form an epitope recognition site. Using these half-antibodies or a vesicle, a viral particle, a composition or a kit thereof, for therapeutic applications, such as the prevention or treatment of cancers, genetic diseases, infectious diseases, and for in vitro diagnostic applications and detecting biological molecules. The half-antibodies include at least two chimeric molecules A and B, each has a polypeptide domain characteristic of a variable domain of a heavy chain or of a light chain of an antibody, and a nucleotide domain, the nucleotide domain of A and that of B being capable of pairing into a double stranded structure. Biologically active nucleic sequences can be grafted onto these chimeric molecules to prevent the expression of target genes in the interior of a human or non-human mammalian cell. 1. A chimeric molecule , termed a “half-antibody” , characterized in that it comprises or consists of two chimeric molecules A and B , each comprising or consisting of:(i) a characteristic polypeptide domain of a variable domain (or VD) of a heavy chain or of a light chain of an antibody, this polypeptide domain being positioned at one end in the chimeric molecules A and B, the polypeptide domain (i) of one of the two chimeric molecules, A or B, being characteristic of a VD of a light chain of an antibody and the polypeptide domain (i) of the other chimeric molecule, respectively B or A, being characteristic of a VD of a heavy chain of an antibody; and(ii) a single stranded nucleotide domain consisting of a polynucleotide, in particular a DNA or a RNA, or an analog of a polynucleotide, in particular a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a methylphosphonate nucleic acid or a thioate nucleic acid, the nucleotide domain of A and that of B being capable of pairing into a double stranded structure, for example in a hydric medium and in particular in a reducing ...

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Номер записи: 15
02-05-2013 дата публикации

Disulfide stabilized antibodies and fragments thereof

Номер: US20130108622A1
Автор: David Paul Humphreys
Принадлежит: UCB PHARMA SA

Disulfide Stabilised Antibodies and Fragments Thereof The present disclosure provides an antibody or antibody fragment comprising at least one Fab molecule, wherein the light chain variable region, V L and the heavy chain region, V B of the Fab molecule are linked by one or more disulfide bonds, and use of the same in treatment or prophylaxis.

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Номер записи: 16
02-05-2013 дата публикации

EXPRESSION OF MONOCLONAL ANTIBODIES IN CILIATE HOST CELLS

Номер: US20130109593A1
Автор: Apelt Jenny, Hartmann Marcus
Принадлежит: CILIAN AG

The present invention is related to a system for the heterologous expression of a monoclonal Antibody (mAb) or a fragment or derivative thereof, said system comprising at least one ciliate host cell, and incorporated, into said ciliate host cell, at least one heterologous nucleic acid molecule encoding for said monoclonal Antibody, or a fragment or derivative thereof. 1. A system for the heterologous expression of a monoclonal Antibody (mAb) or a fragment or derivative thereof , said system comprisinga) at least one ciliate host cell, andb) incorporated, into said ciliate host cell, at least one heterologous nucleic acid molecule encoding for said monoclonal Antibody, or a fragment or derivative thereof.2. The system according to claim 1 , wherein said monoclonal Antibody (mAb) claim 1 , or a fragment or derivative thereof claim 1 , has an N-glycan structure which is essentially fucose-free.3. The system according to claim 1 , wherein said monoclonal Antibody (mAb) claim 1 , or a fragment or derivative thereof claim 1 , has at least one effect selected from the group consisting ofincreased Antibody-Dependent Cellular Cytotoxicity (ADCC),increased Complement-Dependent Cytotoxicity (CDC),increased Antibody-Dependent Apoptosis, and/orincreased Antibody-Dependent Opsonisation.4. The system according to claim 1 , wherein said monoclonal Antibody (mAb) claim 1 , or a fragment or derivative thereof claim 1 , has an extended serum half life.5. The system according to claim 1 , wherein said system further comprisesc) a promoter operably linked to said nucleic acid molecule, and/ord) a signal sequence operably linked to said nucleic acid molecule, which signal sequence accounts for the secretion of the monoclonal Antibody, or the fragment thereof, encoded by the said nucleic acid molecule, into the extracellular medium.6. A vector for the transfection of a ciliate host cell is provided claim 1 , said vector comprising at least one nucleic acid molecule encoding for a ...

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Номер записи: 17
02-05-2013 дата публикации

METHOD OF ALTERING THE BINDING SPECIFICITY OF PLASMA PROTEINS BY OXIDATION-REDUCTION

Номер: US20130109838A1
Автор: MCINTYRE John A.
Принадлежит:

The binding specificity of at least one plasma protein suspended or dissolved in a liquid medium is altered by exposing the protein to an oxidizing agent or an electric current sufficient to alter its binding specificity. A masked protein such as an autoantibody can be recovered from blood or blood products or extracts by oxidizing the protein to change its binding specificity. 1. A method comprising the steps ofproviding a composition comprising at least antibody suspended or dissolved in a liquid medium, the at least one antibody having a binding specificity that can be altered by a change in its redox state, andexposing the composition to an oxidizing agent or DC current sufficient to effect the alteration of the binding specificity of the at least one antibody.2. The method of wherein said liquid medium is diluted whole blood claim 1 , serum claim 1 , or plasma.3. The method of wherein the composition comprises intravenous immunoglobulin (lvlg) suspended or dissolved in a liquid medium.4. The method of wherein the at least one antibody is an antibody of IgG claim 1 , IgA claim 1 , or IgM isotype.5. The method of wherein the at least one antibody is an autoantibody of IgG claim 1 , IgA claim 1 , or IgM isotype. This application is a continuation of U.S. application Ser. No. 12/107,819 filed on Apr. 23, 2008, which is a continuation-in-part application of U.S. application Ser. No. 10/863,365, filed Jun. 9, 2004, now U.S. Pat. No. 7,368,542, which claims the benefit of the filing date of U.S. Provisional Application No. 60/476,607, filed Jun. 9, 2003. U.S. application Ser. No. 12/107,819 is also a continuation of application Ser. No. 11/359,489, filed Feb. 23, 2006, now U.S. Pat. No. 7,892,751, which is a continuation-in-part of U.S. patent application Ser. No. 11/108,826, filed on Apr. 19, 2005, which application is a continuation-in-part of Ser. No. 10/863,365, filed Jun. 9, 2004, now U.S. Pat. No. 7,368,542, which application claims the benefit of U.S. ...

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Номер записи: 18
16-05-2013 дата публикации

MONOMETHYLVALINE COMPOUNDS HAVING PHENYLALANINE SIDE-CHAIN MODIFICATIONS AT THE C-TERMINUS

Номер: US20130123465A1
Автор: Doronina Svetlana O., Kline Toni Beth
Принадлежит: Seattle Genetics, Inc.

Auristatin peptide analogs of MeVal-Val-Dil-Dap-Phe (MMAF) are provided having C-terminal phenylalanine residue side chain replacements or modifications which are provided alone or attached to ligands through various linkers. The related conjugates can target specific cell types to provide therapeutic benefit. 216.-. (canceled)1821.-. (canceled)2427.-. (canceled)29. A compound of claim 28 , having the formula:{'br': None, 'L-LU-D'}or a pharmaceutically acceptable salt or solvate thereof.32. The compound of having the formula:{'br': None, 'sub': 'p', 'Ab-(D).'}33. The compound of claim 31 , wherein the antibody is attached to the drug moiety through a cysteine residue of the antibody.34. The compound of wherein p is 2 to 5.35. The compound of wherein p is 2 to 8.36. (canceled)45. The compound of wherein w is an integer ranging from 2 to 12.46. The compound of wherein w is 2.47. The compound of wherein Wis -valine-citrulline-.48. (canceled)49. (canceled)50. The compound of claim 31 , wherein the antibody is a monoclonal antibody.51. The compound of claim 31 , wherein the antibody is a bispecific antibody.52. The compound of claim 31 , wherein the antibody is a chimeric antibody.53. The compound of claim 31 , wherein the antibody is a humanized antibody.54. The compound of claim 31 , wherein the antibody is an antibody fragment.55. The compound of claim 54 , wherein the antibody fragment is a Fab fragment.56114.-. (canceled) This application claims the benefit of U.S. Provisional Application No. 60/697,767, filed Jul. 7, 2005; the disclosure of which is incorporated by reference herein.The present invention is directed to Drug Compounds, to Drug-Linker-Ligand Conjugates, Drug-Linker Compounds, and Drug-Ligand Conjugates; as well as to compositions including the same, and to methods for using the same to treat cancer, an autoimmune disease, an infectious disease and other pathological conditions. The invention also relates to methods of using Antibody-Drug Conjugate ...

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Номер записи: 19
23-05-2013 дата публикации

CELL CULTIVATION PROCESS

Номер: US20130130316A1
Автор: Joosten Christoph E., Leist Christian, Schmidt Jörg
Принадлежит: Norvartis AG

This invention relates to a cell culture process for the production of polypeptides in mammalian CHO cells characterized by one or more temperature and pH shifts which are adjusted in respect to their timing and step size to reduce cell death, increase product yield and improve product quality. 1. A process for the production of a recombinant polypeptide comprising culturing CHO cells in a medium under conditions comprising at least one temperature shift and at least one pH shift and expressing the recombinant polypeptide , whereinthe cells are grown at a first temperature for at least 3 days and the temperature is then shifted to a second temperature which is between about 1 and about 8° C. lower than the first temperature and the cells are maintained at said second temperature for a period of at least another 2 days;the cells are grown at a first pH value for at least 2 days and the pH is then shifted to a second pH value which is between about 0.05 and about 1 pH units lower than the first pH and the cells are grown at said second pH for at least 1 day.2. The process according to wherein the pH is actively changed between said first and said second pH value.3. The process according to wherein the pH is passively changed between said first and said second pH value.4. The process according to wherein the first temperature is in the range of between about 33° C. and about 38° C.5. The process according to wherein the second temperature is in the range of between about 30° C. and about 37° C.6. The process according to wherein the first pH value is in the range of between about pH 6.8 and about pH 7.5.7. The process according to wherein the second pH value is in the range of between about pH 6.0 and about pH 7.1.8. The process according to wherein said second pH is actively maintained until the end of culturing.9. The process according to wherein the first pH shift is followed by a second pH shift after at least 1 day with the third pH value being about 0.05 pH units ...

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Номер записи: 20
23-05-2013 дата публикации

SINGLE UNIT ANTIBODY PURIFICATION

Номер: US20130131318A1
Автор: Kremer Diderik Reinder, Maarleveld Randal William
Принадлежит:

The present invention relates to a method for the purification of antibodies from a protein mixture produced in a bioreactor, at least comprising the steps of intermediate purification and polishing, wherein the intermediate purification and polishing step comprises in-line anion exchange chromatography (AEX) treatment and hydrophobic interaction chromatography (HIC) treatment in flow through mode. The present invention further relates to a single operational unit comprising both an anion exchange chromatography part and a hydrophobic interaction chromatography part, which are serially connected, wherein the unit comprises an inlet at the upstream end of the anion exchange chromatography part and an outlet at the downstream end of the hydrophobic interaction chromatography part and wherein the unit also comprises an inlet between the anion exchange chromatography part and the hydrophobic interaction chromatography part. 1. Method for the purification of antibodies from a protein mixture produced in a bioreactor , at least comprising the steps of intermediate purification and polishing , wherein the intermediate purification and polishing steps comprise serial in-line anion exchange chromatography (AEX) , yielding as a flow-through fraction a separation mixture , followed by hydrophobic interaction chromatography (HIC) yielding as a flow through fraction a purified antibody preparation , and wherein the purified antibody preparation is subjected to at least one further purification step.2. Method according to wherein anion exchange chromatography and hydrophobic interaction chromatography take place in two separate devices which are serially connected.3. Method according to wherein the serial in-line AEX and HIC are performed as a single unit operation.4. Method according to wherein the separation mixture prior to HIC is supplemented with an adequate amount of lyotropic salt.5. Method according to wherein the separation mixture prior to HIC is supplemented with an ...

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Номер записи: 21
06-06-2013 дата публикации

RAGE Fusion Protein Compositions And Methods Of Use

Номер: US20130142792A1
Автор: Bleck Gregory T., Katragadda Madan, RAJADHYAKSHA Manoj, Rothlein Robert, Violand Bernard N., Webster Jeffrey C., Wentland Jo-Ann
Принадлежит:

Disclosed are fusion proteins comprising a RAGE polypeptide, wherein the RAGE polypeptide comprises a fragment of a mammalian wild type RAGE peptide and at least one point mutation in the RAGE polypeptide portion of the fusion protein relative to the wild type RAGE peptide. The point mutation may remove and/or alter a glycosylation site or an enzyme cleavage site. Also disclosed are nucleic acids encoding such proteins as well as methods of using such proteins for treating RAGE-mediated pathologies. 129-. (canceled)30. A fusion protein comprising a Receptor for Advanced Glycation Endproducts (RAGE) polypeptide linked to an immunoglobulin polypeptide , wherein the RAGE polypeptide comprises a fragment of a mammalian RAGE having a ligand binding domain , and wherein the RAGE fusion protein comprises at least one mutation relative to the wild-type sequence in at least one of the RAGE polypeptide or the immunoglobulin polypeptide , wherein the mutation removes and/or alters at least one of a glycosylation site or an enzyme cleavage site.31. The fusion protein of claim 30 , wherein the RAGE polypeptide is a human RAGE polypeptide.32. The fusion protein of claim 30 , wherein the mutation changes the sequence in the wild-type RAGE polypeptide present at a glycosylation site.33. The fusion protein of claim 30 , wherein the glycosylation site has the amino acid sequence NXS or NXT claim 30 , where X is any amino acid.34. The fusion protein of claim 30 , wherein the mutation changes the sequence in the wild-type RAGE polypeptide from at least one of NIT to QIT claim 30 , or NGS to QGS claim 30 , or NGS to NSS claim 30 , or NST to QST to remove at least one glycosylation site.35. The fusion protein of claim 30 , wherein the glycosylation site is within the ligand binding site or the ligand binding domain of the RAGE polypeptide.36. The fusion protein of claim 30 , wherein the enzyme cleavage site is a furin cleavage site claim 30 , such that the mutation changes the sequence ...

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Номер записи: 22
06-06-2013 дата публикации

FUSION PROTEIN OF EXENDIN-4 AND ITS ANALOG, PREPARATION METHOD AND USE THEREOF

Номер: US20130142795A1
Автор: BAI Xianhong, Li Xianzhong, Tu Hua
Принадлежит: Beijing Dongfang Biotech Co., Ltd.

Provided are a fusion protein of Exendin-4 and its analog, the preparation method and use thereof. The fusion protein is obtained by fusing of Exendin-4 or its analog to Fc region of human IgG2 via a linking peptide, which has the better stability and prolonged serum half-life, and can be used for treating diabetes and obesity. 1. A fusion protein , which is obtained by fusing peptide hormone to transport protein via linker , wherein , the said peptide hormone is Exendin-4 or analogue of Exendin-4 , and the said peptide hormone is capable of lowering the blood glucose; the said transport protein is the Fc fragment of the immunoglobulin IgG2; the said fusion protein is capable of lowering the blood glucose; {'br': None, 'sup': 2', '10', '12', '13', '14', '19', '20', '21', '24', '27', '28', '30', '31', '32', '33', '34', '35', '36', '37', '38', '39, 'His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa-Ser-Xaa-Xaa-Xaa-Glu-Glu-Glu-Ala-Xaa-Xaa-Xaa-Phe-Ile-Xaa-Trp-Leu-Xaa-Xaa-Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa\u2003\u2003Formula I'}, 'the said peptide hormone comprises the sequence shown by Formula IWherein:{'sup': '2', 'Xaais Gly, Thr, Ala, Ser, Leu, Ile or Lys;'}{'sup': '10', 'Xaais Leu, Ala, Ser, Leu, Ile, Glu or Lys;'}{'sup': '12', 'Xaais Lys, Leu, Thr, Ser, Leu, Ile or Cys;'}{'sup': '13', 'Xaais Gln, Thr, Ala, Val, Leu, Ile or Lys;'}{'sup': '14', 'Xaais Met, Tyr, Thr, Ala, Ser, Ile or Lys;'}{'sup': '19', 'Xaais Val, Cys, Ala, Ser, Leu, Ile or Lys;'}{'sup': '20', 'Xaais Arg, Thr, Tyr, Ser, Leu, Ile or Lys;'}{'sup': '21', 'Xaais Leu, Thr, Ala, Asp, Glu, His or Lys;'}{'sup': '24', 'Xaais Glu, Leu, Thr, Ala, Ser, Lys or Ile;'}{'sup': '27', 'Xaais Lys, Ala, Ser, Leu, Thr, Ile or Lys;'}{'sup': '28', 'Xaais Asp, Thr, Ala, Ser, Leu, Ile or Lys;'}{'sup': '30', 'Xaais Gly, Thr, Ala, Ser, Leu, Ile or Arg;'}{'sup': '31', 'Xaais Pro, Val, Ser, Ala, Leu, Ile or Lys;'}{'sup': '32', 'Xaais Ser, Thr, Glu, Ser, Asp, Lys or Ile;'}{'sup': '33', 'Xaais Thr, Ser, Ala, Met, Leu, Ile or Lys ...

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Номер записи: 23
13-06-2013 дата публикации

ANTI-CD137 ANTIBODY AS AN AGENT IN THE TREATMENT OF INFLAMMATORY CONDITIONS

Номер: US20130149301A1
Автор: Meade Harry M.
Принадлежит: GTC Biotherapeutics, Inc.

The present invention relates to the treatment of inflammatory conditions including atherosclerosis and sepsis. In particular, the invention relates to treatment of these conditions using antibodies. 1. A method of treating an inflammatory condition comprising administering to a mammal a therapeutically effective amount of a recombinant aglycosylated CD137 antibody or a prodrug thereof or a pharmaceutically acceptable salt thereof.2. The method of claim 1 , wherein the antibody is aglycosylated in the constant region of the antibody.3. The method of claim 1 , wherein the antibody is aglycosylated in the variable region and the constant region of the antibody.4. The method of claim 1 , wherein the inflammatory condition is sepsis.5. The method of claim 1 , wherein the inflammatory condition is atherosclerosis.6. The method of claim 1 , wherein the recombinant aglycosylated CD137 antibody is encoded by a transgene DNA construct in a bioreactor organism claim 1 , said transgene construct comprising a polypeptide domain.7. The method of claim 6 , wherein the DNA construct is actuated by at least one beta-casein promoter.8. The method of claim 6 , wherein the bioreactor organism is a transgenic ungulate.9. The method of claim 1 , wherein the therapeutically effective amount is about 0.01 mg/kg/day to about 50 mg/kg/day.10. The method of claim 1 , wherein the mammal is a human.11. The method of claim 1 , wherein the aglycosylated CD137 antibody is a chimeric antibody.12. The method of claim 1 , wherein the aglycosylated CD137 antibody is a humanized antibody.13. The method of claim 1 , wherein the aglycosylated CD137 antibody is a fully humanized antibody.14. A method of treating atherosclerosis comprising administering to a human a therapeutically effective amount of an aglycosylated CD137 antibody.15. The method of claim 14 , wherein the therapeutically effective amount is about 0.01 mg/kg.day to about 50 mg/kg/day.16. The method of claim 14 , wherein the CD137 antibody ...

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Номер записи: 24
13-06-2013 дата публикации

SOLUBLE CD80 AS A THERAPEUTIC TO REVERSE IMMUNE SUPRESSION IN CANCER PATIENTS

Номер: US20130149305A1
Автор: OSTRAND-ROSENBERG SUZANNE
Принадлежит:

The present invention provides for a therapeutic cancer treatment using a soluble CD80 fusion protein that binds to PDLL and inhibits PDLL-PD1 interactions thereby overcoming PDLL-induced immune suppression and restoring T cell activation. 1. A method to inhibit and/or reduce binding of PDL1 to PD1 thereby increasing immune response against tumor cells , the method comprising:providing a chimeric polypeptide comprising a CD80 sequence, a modified sequence having 95% homology to the CD80 sequence or a fragment thereof and a Fc of IgG1 sequence; andcontacting a tumor cell with the chimeric polypeptide wherein the chimeric polypeptide binds with PDL1 of the tumor cell.2. The method of claim 1 , wherein the chimeric polypeptide comprises at least two extracellular domains of CD80.3. The method of claim 1 , wherein the CD80 sequence is SEQ ID NO: 4 or a sequence having 95% homology thereof.4. The method of claim 1 , wherein the CD80 sequence consists of two extracellular domains.5. The method of claim 1 , wherein the chimeric polypeptide further comprises a linker positioned between the CD80 sequence and Fc of IgG1; and6. The method of claim 1 , wherein the CD80 sequence is fused directly to the sequence of Fc of IgG1.7. The method of claim 1 , wherein the tumor cell is within a human subject.8. The method of claim 1 , wherein the chimeric polypeptide is combined with a pharmaceutically acceptable carrier.9. A method of blocking of PDL1-PD1 interactions thereby improving T cell activation and reducing tumor progression in a subject claim 1 , the method comprising:providing a chimeric protein comprising at least two extracellular domains of CD80 fused to Fc IgG1 or a sequence of polyneucleotides encoding same; andadministering the chimeric protein or polyneucleotides encoding same to the subject in a therapeutically effective amount to reduce tumor progression in the subject.10. The method of claim 9 , wherein the chimeric protein further comprises a linker positioned ...

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Номер записи: 25
20-06-2013 дата публикации

TRANSGENIC NON-HUMAN ANIMALS FOR PRODUCING CHIMERIC ANTIBODIES

Номер: US20130157354A1
Автор: Kay Robert M., Lonberg Nils
Принадлежит: Genpharm Internationl

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and methods for producing human sequence antibodies which bind to human antigens with substantial affinity. 1. A method for constructing a nucleic acid molecule encoding a human heavy chain immunoglobulin , which method comprises operably linking a nucleic acid molecule encoding a human heavy chain variable region to a nucleic acid molecule encoding a human heavy chain constant region , wherein the human heavy chain variable region is isolated from a B lymphocyte from a transgenic mouse , wherein the genome of said transgenic mouse comprises inactivated murine variable regions and an unrearranged human heavy chain immunoglobulin variable region operably linked to a mu constant region gene segment , wherein the unrearranged heavy chain variable region comprises multiple human VH gene segments , multiple human D gene segments , and multiple human JH gene segments , and wherein the mu constant region is selected from the group consisting of:(a) a human S-mu sequence and a human or mouse mu coding sequence; and(b) a mouse S-mu sequence and a human or mouse mu coding sequence.2. The method of wherein the nucleic acid molecule encoding a human heavy chain immunoglobulin is operably linked to an expression vector.3. The method of wherein the expression vector comprising the nucleic acid molecule encoding a human heavy chain immunoglobulin is introduced into a host cell.4. The method of wherein the host cell is a mammalian cell.5. The method of wherein the mammalian host cell is selected from the group consisting of a CHO cell claim 4 , a SP20 cell claim 4 , and a NS0 cell.6. The method of wherein the human heavy chain immunoglobulin is an IgG claim 1 , IgA claim 1 , or IgE isotype. This Application is a Divisional of copending application Ser. No. 12/239,523, which is a Continuation of application Ser. No. 11/009,873 filed on Dec. 10, 2005, issued as U.S. Pat. No. 7,501,552 ...

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Номер записи: 26
27-06-2013 дата публикации

LIGHT CHAIN-BRIDGED BISPECIFIC ANTIBODY

Номер: US20130165638A1
Автор: Chang Ming-I, Hsu Yu-Shen, Lo Cheng-Kai, Sheu Show-Shan
Принадлежит: DEVELOPMENT CENTER FOR BIOTECHNOLOGY

This invention describes a novel format of monomeric bispecific fusion protein with immune activating property for clinical therapies. A bispecific fusion protein includes a first targeting domain with a specificity for a first target of interest; a bridging domain derived from a constant region of a light chain or heavy chain of an immunoglobulin, which may be a human immunoglobulin; and a second targeting domain with a specificity for a second target of interest. The bispecific fusion protein may further include a linker fused to the N-terminus or the C-terminus of the bridging domain. The first targeting domain is fused to the bridging domain and the second targeting domain is fused to the bridging domain or the linker. The linker may include a GGGGS sequence. The first target of interest may be CD Her/neu, or EpCAM, and the second targeting domain is a T-lymphocyte activating domain. 1. A bispecific fusion protein , comprising:a first targeting domain with a specificity for a first target of interest;a bridging domain derived from a constant region of a light chain or heavy chain of an immunoglobulin; anda second targeting domain with a specificity for a second target of interest.2. The bispecific fusion protein of claim 1 , further comprising a linker fused to the N-terminus or the C-terminus of the bridging domain.3. The bispecific fusion protein of claim 1 , wherein the first targeting domain is fused to the bridging domain or the linker and the second targeting domain is fused to the bridging domain or the linker.4. The bispecific fusion protein of claims 1 , wherein the immunoglobulin is a human immunoglobulin.5. The bispecific fusion protein of claim 4 , wherein the bridging domain is a kappa chain claim 4 , a Lambda chain claim 4 , a Lambda-5 surrogate light chain claim 4 , or a mutant of the kappa chain claim 4 , the Lambda chain claim 4 , or the Lambda-5 surrogate light chain that mimics the light chain constant region claim 4 , or a derivative of the ...

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Номер записи: 27
11-07-2013 дата публикации

Monomeric Polypeptides Comprising Variant FC Regions And Methods Of Use

Номер: US20130177555A1
Автор: Lowe David Christopher, Webster Carl Innes, Wilkinson Ian
Принадлежит: MedImmune Limited

Provided are monomeric polypeptides comprising variant Fc regions and methods of using them. In certain embodiments, monomeric polypeptides of the invention are fusion proteins. In certain embodiments, monomeric polypeptides of the invention are antibodies. 1. A polypeptide comprising IgG immunoglobulin Fc region , wherein the Fc region comprises one or more amino acid substitutions that inhibit dimer formation of the Fc region , wherein the substitutions are at one or more of the following amino acids according to the Kabat EU numbering system: 349 , 351 , 354 , 356 , 357 , 364 , 366 , 368 , 370 , 392 , 394 , 399 , 405 , 407 , 409 , 409 and 439.2. The polypeptide of further comprising a target-specific binding portion selected from the group consisting of:(i) an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region that associate to form the target-specific binding portion;(ii) a domain antibody (dAb); and(iii) a protein scaffold.3. (canceled)4. The polypeptide of claim 1 , wherein said polypeptide is a fusion protein comprising an immunoglobulin Fc region fused to a therapeutic polypeptide.57-. (canceled)8. The polypeptide of claim 1 , wherein the one or more amino acids are substituted with an amino acid selected from the group consisting of:(i) an amino acid having a positively charged side chain;(ii) an amino acid having a negatively charged side chain;(iii) an amino acid having a hydrophilic side chain; and(iv) an amino acid having a large side chain.9. (canceled)10. The polypeptide according to claim 1 , wherein the Fc region is from a human IgG immunoglobulin or a mouse IgG immunoglobulin.11. (canceled)12. The polypeptide according to claim 10 , wherein the Fc region is from an IgG1 claim 10 , IgG2 claim 10 , IgG3 or IgG4 immunoglobulin.13. (canceled)14. The polypeptide according to claim 1 , wherein one or more of the following amino acid positions have been substituted with an amino acid having a positively charged ...

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Номер записи: 28
11-07-2013 дата публикации

PROCESS FOR PURIFYING PROTEINS

Номер: US20130178607A1
Автор: WILD GAVIN BARRY
Принадлежит: UCB PHARMA S.A.

The present invention provides method for purifying a recombinant protein from a gram-negative bacterial host cell sample or extract thereof wherein said host cell expresses a recombinant protein and a recombinant disulphide isomerase DsbC; comprising: a. adjusting the pH of the host cell sample or extract thereof to a pH of 5 or less to precipitate the recombinant disulphide isomerase; and b. separating precipitated recombinant disulphide isomerase DsbC from the recombinant protein to produce a recombinant protein sample. 1. A method for purifying a recombinant protein from a gram-negative bacterial host cell sample or extract thereof wherein said host cell expresses a recombinant protein and a recombinant disulphide isomerase DsbC; wherein the method comprises:a) adjusting the pH of the host cell sample or extract thereof to a pH of 5 or less to precipitate the recombinant disulphide isomerase; andb) separating precipitated recombinant disulphide isomerase from the recombinant protein to produce a recombinant protein sample.2. The method according to claim 1 , wherein the pH of the host cell sample or extract thereof is adjusted to a pH of 4.5 or less.3. The method according to claim 2 , wherein the pH of the host cell sample or extract thereof is adjusted to a pH of 4.5 to 3.0.4. The method according to claim 2 , wherein the pH of the host cell sample or extract thereof is adjusted to a pH of 3 or less.5. The method according to claim 4 , wherein in step a) host cell dipeptide binding protein is further precipitated and in step b) host cell dipeptide binding protein is further separated from the recombinant protein.6. The method according to claim 1 , wherein the disulphide isomerase comprises a histidine tag.7E. coli.. The method according to claim 1 , wherein the gram-negative bacterial host cell is is8. The method according to claim 1 , wherein the host cell comprises Fkpa and/r Skp.9. The method according to claim 1 , wherein in step a) the pH of the host ...

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Номер записи: 29
18-07-2013 дата публикации

SUPER-HUMANIZED ANTIBODIES

Номер: US20130184440A1
Автор: Thullier Philippe
Принадлежит: ETAT FRANÇAIS (MINISTÈRE DE LA DÉFENSE), SERVICE DE SANTÉ DES ARMÉES

A method of preparing a germinalized hypervariable antibody region directed against a target, as well as the antibodies, antibody fragments, vectors and compositions including the germinalized hypervariable region. 1. A method of preparing a germinalized hypervariable antibody region directed against a target , comprising the following steps:a) obtaining the peptide sequence of a hypervariable region of a mammalian antibody or antibody fragment, with the exception of a human IgM, said mammal having a human homologous sequence, and said antibody or antibody fragment being directed against said target;b) comparing the peptide sequence of the hypervariable region obtained in a) with the peptide sequences encoded by the human germline genes V, (D), J, in order to identify at least one human germline gene V, (D) or J coding for the peptide sequence closest to the peptide sequence of the hypervariable region obtained in a);c) for each amino acid different between the peptide sequence obtained in a) and the closest peptide sequence obtained in b), substitution by directed mutagenesis in vitro of each amino acid of the peptide sequence of the hypervariable region obtained in a), with the corresponding amino acid of the peptide sequence encoded by the human germline gene V, (D) or J identified in b), in order to obtain a series of mutated mammalian hypervariable regions, each peptide sequence of the hypervariable region comprising at least one mutation;d) selecting the mutated hypervariable regions obtained in c) having an affinity for said target comparable to or greater than that of the hypervariable region of the antibody or of the antibody fragment obtained in a); ande) optionally, preparing the peptide sequence of the hypervariable region of antibody or of antibody fragment directed against said target comprising some or all of the mutations present in at least one peptide sequence of the selection obtained in d).2. The method as claimed in claim 1 , characterized in ...

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Номер записи: 30
25-07-2013 дата публикации

Multimeric Proteins Comprising Immunoglobulin Constant Domains

Номер: US20130189247A1
Автор: Bramhill David, Dimitrov Dimiter S., Gehlsen Kurt R., Gong Rui
Принадлежит: RESEARCH CORPORATION TECHNOLOGIES, INC.

The present invention relate to small binding proteins comprising two or more protein domains derived from a CH2 domain or CH2-like domain of an immunoglobulin in which the CH2 domains have been altered to recognize one or more target proteins and, in some embodiments, retain, or have modified, certain secondary effector functions. 1. A recombinant CH2 domain (CH2D) multimer comprising a first immunoglobulin CH2 domain linked to a second immunoglobulin CH2 domain , either or both CH2 domains are stabilized and modified compared to a wild-type CH2 domain , at least one CH2 domain comprises at least one structural loop modified to an antigen-binding loop.2. (canceled)3. The CH2D multimer of claim 1 , wherein the first immunoglobulin CH2 domain and a second immunoglobulin CH2 domain are linked via a linker.4. The CH2D multimer of claim 3 , wherein the linker comprises a peptide between about 5 to 20 amino acids in length.5. The CH2D multimer of claim 3 , wherein the linker comprises at least one multimerizing domain.6. The CH2D multimer of claim 3 , wherein the linker is a hinge component.712-. (canceled)13. The CH2D multimer of further comprising a third immunoglobulin CH2 domain.14. The CH2D multimer of further comprising a fourth immunoglobulin CH2 domain.1516-. (canceled)17. The CH2D multimer of claim 1 , wherein an N-terminus of the first immunoglobulin CH2 domain is linked to a C-terminus of the second immunoglobulin CH2 domain.18. The CH2D multimer of claim 1 , wherein an N-terminus of the second immunoglobulin CH2 domain is linked to a C-terminus of the first immunoglobulin CH2 domain.19. The CH2D multimer of claim 1 , wherein a C-terminus of the first immunoglobulin CH2 domain is linked to a C-terminus of the second immunoglobulin CH2 domain.20. The CH2D multimer of claim 1 , wherein an N-terminus of the first immunoglobulin CH2 domain is linked to an N-terminus of the second immunoglobulin CH2 domain.21. (canceled)22. The CH2D multimer of wherein the at least ...

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Номер записи: 31
25-07-2013 дата публикации

DIPEPTIDES TO ENHANCE YIELD AND VIABILITY FROM CELL CULTURES

Номер: US20130189737A1
Автор: Deshpande Rohini, Kang Sohye, McCoy Rebecca E., MIRANDA Leslie P., Morris Arvia E.
Принадлежит: Amgen Inc.

The present invention relates to the culture of animal cells in serum-free culture medium. The present invention provides particular dipeptides that can improve recombinant protein production and cell viability in such cultures, especially in the absence of peptones. 1. A method of culturing Chinese hamster ovary (CHO) cells that have been recombinantly engineered to express a protein , the method comprising growing the CHO cells in a serum-free medium during a growth phase , and growing the CHO cells in a serum-free defined production medium during a production phase , wherein during the production phase the serum-free medium is supplemented with at least one dipeptide selected from Tyr-His , Tyr-Lys , Tyr-Ala , Tyr-Val , His-Gly , and Ala-His , and wherein the titer of the protein is improved in the presence of the dipeptide or dipeptides as compared to the absence of the dipeptide or dipeptides.2. The method of claim 1 , wherein the dipeptide is added at a final concentration in the serum-free defined production medium from about 0.1 g/L to about 5 g/L.3. The method of claim 1 , wherein the dipeptide is added in a feed medium to the production phase.4. The method of claim 1 , wherein at least two dipeptides are added.5. The method of claim 4 , wherein one dipeptide is Thr-Phe claim 4 , His-Glu claim 4 , Glu-His claim 4 , His-Ser or His-Gln.6. The method of claim 5 , wherein the dipeptides comprise Tyr-His and Thr-Phe.7. The method of claim 1 , wherein the serum-free defined production medium contains putrescine and/or spermine.8. The method of claim 1 , wherein the serum-free defined production medium contains insulin-like growth factor type 1 (IGF-1).9. The method of claim 1 , wherein the protein is a human antibody claim 1 , a humanized antibody claim 1 , a chimeric antibody claim 1 , a recombinant fusion protein claim 1 , or a cytokine.10. A cell culture comprising a Chinese hamster ovary (CHO) cell line recombinantly engineered to express a protein claim 1 , ...

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Номер записи: 32
01-08-2013 дата публикации

PROTEIN PRODUCTION

Номер: US20130197196A1
Автор: Becker Eric, FLORIN Lore, Fugmann Tim, Hausser Angelika, KAUFMANN Hitto, Olayioye Monilola
Принадлежит: Boehringer Ingelheim Pharma GmbH & Co. KG

The invention concerns the field of protein production and cell culture technology. CERT is identified as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT towards its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. The present invention shows that CERT in turn is critical for PKD activation and PKD dependent protein cargo transport to the plasma membrane. The interdependence of PKD and CERT is thus a key to the maintenance of Golgi membrane integrity and secretory transport. 138-. (canceled)3967-. (canceled)68. A protein of interest or an antibody produced in a cell by a method comprising:a. increasing the expression or activity of a protein having an amino acid sequence comprising a steroidogenic acute regulatory related lipid transfer (START) domain or a derivative or mutant thereof; andb. effecting the expression of said protein of interest.6975-. (canceled) This application is a Divisional application of U.S. application Ser. No. 12/040,198, which claims priority benefit from EP 07103406.0, filed Mar. 2, 2007, EP 07104226.1, filed Mar. 15, 2007, EP 07116358.8, filed Sep. 13, 2007, and U.S. Provisional Patent Application No. 60/893,025, filed Mar. 5, 2007, and the contents of which are all incorporated herein.1. Technical FieldThe invention concerns the field of cell culture technology. It concerns a method for producing proteins as well as a method to generate novel expression vectors and host cells for biopharmaceutical manufacturing. The invention further concerns pharmaceutical compositions and methods of treatment.2. BackgroundThe market for biopharmaceuticals for use in human therapy continues to grow at a high rate with 270 new biopharmaceuticals being evaluated in clinical studies and estimated sales of 30 billions in 2003 (Werner, 2004). Biopharmaceuticals can be produced from various host cell ...

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Номер записи: 33
01-08-2013 дата публикации

MICE EXPRESSING A LIMITED IMMUNOGLOBULIN LIGHT CHAIN REPERTOIRE

Номер: US20130198880A1
Автор: BABB Robert, BUCKLER David R., DAVIS Samuel, MACDONALD Lynn, MCWHIRTER John, MEAGHER Karolina A., MURPHY Andrew J., STEVENS Sean
Принадлежит: Regeneron Pharmaceuticals, Inc.

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that present a choice of two human light chain variable gene segments such that the immunoglobulin light chains expresses by the mouse comprise one of the two human light chain variable gene segments. Methods for making bispecific antibodies having universal light chains using mice as described herein, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, and host cells are provided. 1. A mouse comprising{'sub': L', 'L, 'no more than two human Vgene segments and two or more human Jgene segments operably linked to a mouse or rat light chain constant region; and'}{'sub': H', 'H', 'H, 'one or more human V, one or more human D, and one or more human Jgene segments operably linked to a non-human constant region;'}{'sub': 'L', 'wherein the human gene segments are capable rearranging and encoding human variable domains of an antibody, and further wherein the mouse does not comprise an endogenous Vgene segment that is capable of rearranging to form an immunoglobulin light chain.'}2. The mouse of claim 1 , wherein the light chain constant region is a rat Cκ region.3. The mouse of claim 1 , wherein the light chain constant region is a mouse Cκ region.4. The mouse of claim 1 , wherein the mouse comprises five human Jgene segments.5. The mouse of claim 4 , wherein the five human Jgene segments are human Jκ1 claim 4 , Jκ1 Jκ3 claim 4 , Jκ4 and Jκ5.6. The mouse of claim 1 , wherein the no more than two human Vgene segments are selected from a human Vκ1-39 gene segment claim 1 , a human Vκ3-20 gene segment claim 1 , and a combination thereof.7. The mouse of claim 6 , wherein the no more than two human Vgene segments are a human Vκ1-39 gene segment and a human Vκ ...

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Номер записи: 34
08-08-2013 дата публикации

Composition for the Anti-Cancer Metastasis Containing DLK1-Fc Fusion Protein as an Effective Ingredient

Номер: US20130202592A1
Автор: Jo Kiwon, Kim Eun Jin, Lee Donghee, Park Chan-Woong, Park Ji Hyun, Park Young Woo, Park Yun Jung, Yu Jung
Принадлежит: Korea Research Institute of Bioscience and Biotechnology

A recombinant expression vector, comprising extracellular soluble domain genes of DLK1 and IgG antibody Fc domain genes, is constructed, and DLK1-Fc fusion protein is expressed and purified at 293E cell. The invention confirmed the efficacy as a drug for inhibiting cancer metastasis by confirming markedly reduced migration of cancer cells by DLK1-Fc fusion protein and also computing pharmacokinetic parameters. DLK1-Fc fusion protein has relatively higher stability than non-fusion protein, significantly reduces migration of various cancer cell lines, and provides superior cancer metastasis inhibition effect even at small concentration. Accordingly, DLK1-Fc fusion protein can be used efficaciously as an effective ingredient of a composition for inhibiting cancer metastasis. 1. An extracellular soluble domain of DLK1 (delta-like 1 homolog).2. The extracellular soluble domain of DLK1 according to claim 1 , which is 200-300 amino acids in size.3. The extracellular soluble domain of DLK1 according to claim 1 , comprising an amino acid sequence of SEQ ID NO:4.4. A DLK1-Fc fusion protein comprising an extracellular soluble domain of DLK1 and a human antibody Fc domain.5. A polynucleotide coding the DLK1-Fc fusion protein of .6. A recombinant vector containing the polynucleotide of .7. A recombinant cell line in which the recombinant vector of is transfected into a host cell.8. A method for preparing a DLK1-Fc fusion protein claim 6 , comprising:{'claim-ref': {'@idref': 'CLM-00007', 'claim 7'}, '1) culturing the recombinant cell line of ; and'}2) separating DLK1-Fc fusion protein from cell line culture.910-. (canceled)11. A method for inhibiting cancer metastasis claim 6 , comprising a step of administering a pharmaceutically effective amount of the extracellular soluble domain of DLK1 of or the DLK1-Fc fusion protein of into a subject with a metastatic cancer.12. The method according to claim 11 , wherein the cancer is one selected from a group consisting of skin cancer ...

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Номер записи: 35
08-08-2013 дата публикации

IMMUNOGLOBULIN G FC REGION BINDING POLYPEPTIDE

Номер: US20130203962A1
Автор: Abrahmsén Lars
Принадлежит: GE HEALTHCARE BIO-SCIENCES AB

An immunoglobulin G Fc region binding polypeptide is provided, consisting of an amino acid sequence selected from i) and an amino acid sequence which has at least 95% identity thereto. Also provided are methods for producing the polypeptide, compositions comprising the polypeptide, polynucleoties encoding the polypeptide, multimers of the polypeptide, and methods of using the polypeptide. 2. The IgG Fc-binding polypeptide of claim 1 , wherein [spacer1] is selected from A claim 1 , AE and AEA.3. The IgG Fc-binding polypeptide of claim 2 , wherein [spacer1] is A.4. The IgG Fc-binding polypeptide of claim 1 , wherein Xis H.5. The IgG Fc-binding polypeptide of claim 1 , wherein Xis any amino acid residue except for N claim 1 , for example T.6. The IgG Fc-binding polypeptide of claim 1 , wherein Xis N.7. The IgG Fc-binding polypeptide of claim 1 , wherein Xis selected from E and A claim 1 , for example E.8. The IgG Fc-binding polypeptide of claim 1 , wherein Xis any amino acid residue except for N claim 1 , for example D.9. The IgG Fc-binding polypeptide of claim 1 , wherein Xis N.10. The IgG Fc-binding polypeptide of claim 1 , wherein all amino acid residues of [spacer2] are independently selected from A claim 1 , E claim 1 , F claim 1 , G claim 1 , I claim 1 , K claim 1 , L claim 1 , P claim 1 , Q claim 1 , R claim 1 , S claim 1 , T and V claim 1 , in particular from A claim 1 , E claim 1 , G claim 1 , K claim 1 , P claim 1 , Q claim 1 , R claim 1 , S and T claim 1 , such as from G claim 1 , Q and S.11. The IgG Fc-binding polypeptide of claim 1 , wherein [spacer2] is G.12. The IgG Fc-binding polypeptide of claim 1 , wherein n is 1.13. The IgG Fc-binding polypeptide of claim 1 , wherein n is 2.14. The IgG Fc-binding polypeptide of claim 1 , wherein n is 4.15. The IgG Fc-binding polypeptide of claim 1 , which binds to IgG Fc such that the Kvalue of the interaction is at most 1×10M.16. The IgG Fc-binding polypeptide of claim 15 , which binds to IgG Fc such that the Kvalue ...

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Номер записи: 36
08-08-2013 дата публикации

USE OF SMALL MOLECULES IN METHODS FOR PURIFICATION OF BIOMOLECULES

Номер: US20130203969A1
Автор: Dupont Alison, Jaber Jad, Kozlov Mikhail, Moya Wilson, Potty Ajish, Stone Matthew T.
Принадлежит: EMD MILLIPORE CORPORATION

The present invention relates to novel and improved methods for the purification of biomolecules. In particular, the present invention relates to methods of protein purification which employ small molecules, which include at least one non-polar group and at least one cationic group or which include at least one non-polar group and at least one anionic group. 1. A method of separating a target biomolecule from one or more insoluble impurities in a sample: the method comprising the steps of:(i) providing a sample comprising a biomolecule of interest and one or more insoluble impurities;(ii) contacting the sample with a small molecule comprising at least one cationic group and at least one non-polar group, in an amount sufficient to form a precipitate comprising the one or more insoluble impurities; and(iii) removing the precipitate from the sample, thereby to separate the target molecule from the one or more insoluble impurities.2. The method of claim 1 , where the non-polar group is aromatic.3. The method of claim 1 , wherein the non-polar group is aliphatic.4. The method of claim 1 , wherein the one or more insoluble impurities are selected from whole cells and cell debris.5. The method of claim 1 , wherein the small molecule is selected from the group consisting of a monoalkyltrimethyl ammonium salt claim 1 , a monoalkyldimethylbenzyl ammonium salt claim 1 , a dialkyldimethyl ammonium salt claim 1 , a heteroaromatic ammonium salt claim 1 , a polysubstituted quaternary ammonium salt and a bis-quaternary ammonium salt.6. The method of claim 5 , wherein a monoalkyltrimethyl ammonium salt is selected from the group consisting of cetyltrimethylammonium bromide claim 5 , cetyltrimethylammonium chloride claim 5 , tetradecyltrimethylammonium bromide claim 5 , tetradecyltrimethylammonium chloride claim 5 , alkyltrimethyl ammonium chloride claim 5 , alkylaryltrimethyl ammonium chloride claim 5 , dodecyltrimethylammonium bromide claim 5 , dodecyltrimethylammonium chloride ...

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Номер записи: 37
15-08-2013 дата публикации

POLYPEPTIDES HAVING ANTIVIRAL ACTIVITY AND METHODS FOR USE THEREOF

Номер: US20130209464A1
Автор: Husk Adam, Matoba Nobuyuki, Sankaran Sudha
Принадлежит: UNIVERSITY OF LOUISVILLE RESEARCH FOUNDATION, INC

A polypeptide is provided that comprises an actinohivin variant polypeptide having an amino acid sequence selected from SEQ ID NOS: 4-12. The polypeptide can be provided as part of a fusion protein that includes the actinohivin variant polypeptide and either a fragment crystallizable domain of an antibody (Fc), a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv). Isolated nucleic acid molecules encoding the polypeptides are also provided along with vectors and plant cells capable of expressing the polypeptides. Methods of treating an infection of a subject by an enveloped virus are further provided and include administering an effective amount of the polypeptides to a subject. 1. An isolated polypeptide , comprising an actinohivin variant polypeptide having an amino acid sequence selected from SEQ ID NOS: 4-12.2. The polypeptide of claim 1 , wherein the actinohivin variant polypeptide comprises the sequence of SEQ ID NO: 4.3. The polypeptide of claim 1 , further comprising a second polypeptide selected from: a fragment crystallizable domain of an antibody (Fc); a fragment antigen-binding domain of an antibody (Fab); and a single chain variable fragment of an antibody (scFv) claim 1 , wherein the actinohivin variant polypeptide and the second polypeptide comprise a fusion protein.4. The polypeptide of claim 3 , wherein the antibody is a monoclonal antibody.5. The polypeptide of claim 3 , wherein the second polypeptide is Fab.6. The polypeptide of claim 3 , wherein the second polypeptide is scFv.7. The polypeptide of claim 3 , wherein the second polypeptide is Fc.8. The polypeptide of claim 3 , wherein the polypeptide further comprises a peptide linker for connecting the actinohivin variant polypeptide to the second polypeptide.9. The polypeptide of claim 3 , wherein the peptide linker comprises an amino acid sequence of SEQ ID NO: 13.10. The polypeptide of claim 1 , wherein the actinohivin variant ...

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Номер записи: 38
15-08-2013 дата публикации

ENHANCED PROTEIN EXPRESSION

Номер: US20130210075A1
Автор: Satakarni Makkapati, Satyam Subrahmanyam, Vaibhav S. Nikam
Принадлежит:

The application describes methods to enhance protein production using mammalian cells. Increased production of heterologous protein can be obtained by increasing osmolality and lowering temperature. The method maintains the cell growth rate and provides high product yield. 1. A cell culture process for protein expression in host cells comprising:a) growing cells at one osmolar value and temperature;b) subjecting cells to at least one higher osmolar value and temperature reduction; andc) recovering the protein from the culture.2. A process according to claim 1 , wherein the cells are grown in step a) in an osmolality range from about 320-410 mOsm/kg.3. A process according to claim 1 , wherein cells in step b) are subjected to an osmolality range from about 380-450 m Osm/kg.4. A process according to claim 1 , wherein cells in step b) are subjected to an osmolality range greater than 450 mOsm/kg but less than 550 mOsm/kg.5. A process according to claim 1 , wherein cells in step b) are subjected to more than one higher osmolar value.6. A cell culture process for protein expression in host cells comprising:a) growing cells at an initial osmolar value and temperature;b) culturing cells in at least one higher osmolar value;c) subjecting cells to at least one increased osmolar value and reduction in temperature; andd) recovering the protein from the culture.7. A process according to claim 6 , wherein cells are grown in step a) at an osmolality about 310-330 mOsm/kg.8. A process according to claim 6 , wherein the cells in step b) are cultured in an osmolality range from about 320-410 mOsm/kg.9. A process according to claim 6 , wherein the cells in step c) are subjected to an osmolality range greater than 450 mOsm/kg and less than 550 mOsm/kg.10. A process according to claim 6 , wherein at least one osmolality change is obtained by incremental addition of nutrients.11. A cell culture process for expression of protein from host cells comprising:a) growing cells at an initial ...

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Номер записи: 39
15-08-2013 дата публикации

Methods of Modifying Eukaryotic Cells

Номер: US20130210137A1
Автор: MURPHY Andrew J., YANCOPOULOS George D.
Принадлежит: Regeneron Pharmaceuticals, Inc.

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification. 1. A genetically modified mouse that expresses an antibody that comprises a human immunoglobulin sequence , wherein the human immunoglobulin sequence is derived from a human gene segment at a modified endogenous mouse immunoglobulin locus in the germline of the mouse.2. The genetically modified mouse of claim 1 , wherein the antibody derived from the modified endogenous mouse immunoglobulin locus in the germline of the mouse comprises a human heavy chain variable domain sequence.3. The genetically modified mouse of claim 1 , wherein the antibody derived from the modified endogenous mouse immunoglobulin locus in the germline of the mouse comprises a human light chain variable domain sequence.4. The genetically modified mouse of claim 3 , wherein the antibody derived from the modified endogenous mouse immunoglobulin locus in the germline of the mouse comprises a human λ light chain variable domain sequence.5. The genetically modified mouse of claim 3 , wherein the antibody derived from the modified endogenous mouse immunoglobulin locus in the germline of the mouse comprises a human κ light chain variable sequence.6. The genetically modified mouse of claim 2 , wherein the antibody derived from the modified endogenous mouse immunoglobulin locus in the germline of the mouse ...

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Номер записи: 40
29-08-2013 дата публикации

VIRAL CHEMOKINE-ANTIGEN FUSION PROTEINS

Номер: US20130224199A1
Автор: Arya Bira, Kwak Larry W.
Принадлежит:

The present invention relates to a vaccine for increasing the immunogenicity of a tumor antigen thus allowing treatment of cancer, as well as a vaccine that increases the immunogenicity of a viral antigen, thus allowing treatment of viral infection, including immunodeficiency virus (HIV) infection. In particular, the present invention provides a fusion protein comprising a viral chemokine fused to either a tumor antigen or viral antigen which is administered as either a protein or nucleic acid vaccine to elicit an immune response effective in treating cancer or effective in treating or preventing viral infection. 1. A fusion polypeptide comprising a viral chemokine and a human tumor antigen , wherein the viral chemokine comprises viral macrophage inflammatory protein III (vMIPIII).2. The fusion polypeptide of claim 1 , wherein the human tumor antigen comprises a B cell tumor antigen.3. The fusion polypeptide of claim 2 , wherein the B cell tumor antigen is selected from the group consisting of:(1) an antibody produced by a B cell tumor;(2) a single chain antibody comprising linked VH and VL domains which retain the conformation and specific binding activity of the native idiotype of the antibody produced by a B cell tumor; and(3) an epitope of an idiotype of an antibody produced by a B cell tumor.4. The fusion polypeptide of claim 3 , wherein the B cell tumor antigen comprises sFv38.5. The fusion polypeptide of claim 1 , wherein the human tumor antigen comprises gp100.6. The fusion polypeptide of claim 1 , wherein the human tumor antigen comprises Muc-1.7. The fusion polypeptide of claim 1 , wherein the human tumor antigen comprises the amino acid sequence of SEQ ID NO: 1.8. A composition comprising the fusion polypeptide of in a pharmaceutically acceptable carrier.9. A method of producing an immune response in a subject claim 8 , comprising administering to the subject the composition of .10. The method of claim 9 , wherein the immune response is an effector T cell ...

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Номер записи: 41
05-09-2013 дата публикации

Cell Display Of Antibody Libraries

Номер: US20130231463A1
Автор: GAO CHANGSHOU, WU HERREN
Принадлежит: MEDIMMUNE, LLC

The present invention relates to a viral vector encoding for a library of antibodies or antibody fragments that are displayed on the cell membrane when expressed in a cell. The present invention provides cells comprising the viral vector nucleic acids and methods of screening the libraries for antibodies or antibody fragments with desired characteristics. 120-. (canceled)21. An isolated antibody comprising a human IgG1 Fc region having a reduced affinity for an effector molecule wherein said human IgG1 Fc region comprises an amino acid insertion after an amino acid residue at a position selected from the group consisting of: 230 , 231 , 232 , 233 , 234 , 235 , 236 , 237 , 238 , 239 and 240 , wherein the numbering is according to EU index as set forth in Kabat.22. The antibody of claim 21 , wherein the effector molecule is selected from the group consisting of C1q claim 21 , FcγRI claim 21 , FcγRII and FcγRIIIA.23. The antibody of claim 21 , wherein the antibody has reduced effector function.24. The antibody of claim 23 , wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).25. The antibody of claim 21 , wherein the human IgG1 Fc region has a reduced affinity for C1q claim 21 , FcγRI claim 21 , FcγRII and FcγRIIIA.26. The antibody of claim 25 , wherein the effector function is complement dependent cytotoxicity (CDC).27. An Fc fusion protein comprising a human IgG1 Fc region having a reduced affinity for an effector molecule wherein said human IgG1 Fc region comprises an amino acid insertion after an amino acid residue at a position selected from the group consisting of: 230 claim 25 , 231 claim 25 , 232 claim 25 , 233 claim 25 , 234 claim 25 , 235 claim 25 , 236 claim 25 , 237 claim 25 , 238 claim 25 , 239 and 240 claim 25 , wherein the numbering is according to EU index as set forth in Kabat.28. The Fc fusion protein of claim 27 , wherein the effector molecule is selected from the group consisting of C1q claim 27 , FcγRI claim 27 , ...

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Номер записи: 42
12-09-2013 дата публикации

IMMUNOGLOBULIN Fc FRAGMENT TAGGING ACTIVATION OF ENDOGENOUS CD4 AND CD8 T CELLS AND ENHANCEMENT OF ANTITUMOR EFFECTS OF LENTIVECTOR IMMUNIZATION

Номер: US20130236456A1
Автор: He Yukai, MUNN DAVID H.
Принадлежит: Georgia Health Sciences University Research Institute, Inc.

A lentivector has been engineered to express a fusion antigen composed of hepatitis B surface protein (HBsAg) and IgG2a Fc fragment (HBS-Fc-Iv) to increase both the magnitude of CD8 response and to induce effective co-activation of CD4 T cells. Immunization with this HBS-Fc-Iv caused significant regression of established tumors. Immunological analysis revealed that, compared to HBS-Iv without the Fc fragment, immunization with HBS-Fc-Iv markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor, while substantially decreasing the Treg ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-Iv immunization were dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of the CD4 T cells from the HBS-Fc-Iv immunized mice could activate endogenous CD8 T cells in an IFNγ-dependent manner. Endogenous CD4 T cells can be activated by lentivirus expressing Fc-tagged antigen to provide another layer of help, i.e., creating a Th1/Tc1 like pro-inflammatory milieu within the tumor lesion to help the effector phase of immune responses to enhance the antitumor effect. 1. A recombinant fusion protein for potentiating an immune system , the fusion protein comprising a self-assembling virus-like particle-forming polypeptide; an Fcγ receptor (FcγR)-binding ligand polypeptide , and a target antigen polypeptide , thereby forming a tripartite fusion polypeptide.2. The recombinant fusion protein of claim 1 , wherein the target antigen polypeptide is positioned between the self-assembling virus-like particle-forming polypeptide and the Fcγ receptor (FcγR)-binding ligand polypeptide.3. The recombinant fusion protein of claim 1 , wherein the self-assembling virus-like particle-forming polypeptide is selected from the group consisting of: an HPV L1 protein claim 1 , an HPV L2 protein claim 1 , an influenza Hemagglutinin A claim 1 , an influenza ...

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Номер записи: 43
12-09-2013 дата публикации

RECONSTITUTED POLYPEPTIDES

Номер: US20130237684A1
Автор: KOIDE Shohei
Принадлежит:

The present invention provides modified fibronectin type III (Fn3) molecules, and nucleic acid molecules encoding the modified Fn3 molecules. Also provided are methods of preparing these molecules, and kits to perform the methods. 1. A fibronectin monobody polypeptide comprising a first fibronectin monobody fragment having at least two beta-strand domains with a loop region linked between each beta-strand domain , and at least one additional fibronectin monobody fragment having at least two beta strand domains with a loop region linked between each beta-strand domain.2. The monobody of claim 1 , wherein the first fibronectin monobody fragment is a fibronectin type III (Fn3) polypeptide claim 1 , a fibronectin type II (Fn2) polypeptide claim 1 , or a fibronectin type I (Fn1) polypeptide.3. The monobody of claim 1 , wherein the additional fibronectin monobody fragment is a fibronectin type III (Fn3) polypeptide claim 1 , a fibronectin type II (Fn2) polypeptide claim 1 , or a fibronectin type I (Fn1) polypeptide.4. The monobody of claim 1 , wherein the first and the additional fibronectin monobody fragments are fibronectin type III (Fn3) fragments.5. The monobody of claim 1 , wherein the first fibronectin monobody fragment is a fibronectin type III (Fn3) fragment and the additional fibronectin monobody fragment is a fibronectin type II (Fn2) fragment.6. The monobody of claim 1 , wherein at least one loop region is capable of binding to a specific binding partner (SBP) to form a polypeptide:SBP complex.7. The monobody of claim 1 , wherein at least one of the loop regions of the first or additional fibronectin monobody fragments comprises amino acid residues: i) from 15 to 16 inclusive in an AB loop; ii) from 22 to 30 inclusive in a BC loop; iii) from 39 to 45 inclusive in a CD loop; iv) from 51 to 55 inclusive in a DE loop; v) from 60 to 66 inclusive in an EF loop; or vi) from 76 to 87 inclusive in an FG loop.8. A fibronectin monobody polypeptide comprising a first ...

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Номер записи: 44
19-09-2013 дата публикации

Transgenic Animals

Номер: US20130243759A1
Автор: Friedrich Glenn A., Lee E-Chiang
Принадлежит: KYMAB LTD.

The present invention relates inter alia to fertile non-human vertebrates such as mice and rats useful for producing antibodies bearing human variable regions, in which endogenous antibody chain expression has been inactivated. 1. A method of making a mouse cell or a fertile mouse that is homozygous for a transgenic antibody heavy chain locus ,the mouse having a genome that(a) comprises each transgenic heavy chain locus on a respective copy of chromosome 12; and(b) is inactivated for endogenous antibody heavy chain expression;the method comprising the steps of(c) constructing a transgenic mouse embryonic stem cell (ES cell) comprising a transgenic antibody heavy chain locus by inserting one or more human VH gene segments, one or more human D gene segments and one or more human JH gene segments into DNA of a chromosome 12 so that the human gene segments are operably connected upstream of a mouse or human heavy chain constant region;(d) simultaneously or separately from step (c), deleting all or part of the mouse endogenous heavy chain VDJ region of said chromosome 12 to inactivate endogenous antibody heavy chain expression, wherein the deletion includes mouse ADAM6 coding sequences;(e) simultaneously or separately from step (c) or (d), inserting into the ES cell genome one or more ADAM6 coding sequences.2. The method of claim 1 , wherein said heavy chain constant region comprises one of both of Cmu and Cgamma.3. The method of claim 1 , further comprising the step of f) developing the ES cell into a fertile mouse or a progeny thereof whose genome is homozygous for said transgenic heavy chain locus and encodes ADAM6 claim 1 , wherein all or part of the endogenous heavy chain VDJ region has been deleted from both chromosomes 12 in the genome.4. The method of claim 1 , wherein the mouse is a male.5. A method of making a mouse cell or a fertile mouse that is homozygous for a transgenic antibody heavy chain locus claim 1 ,the mouse having a genome that(a) comprises each ...

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Номер записи: 45
19-09-2013 дата публикации

METHOD OF PRODUCING ANTIBODIES

Номер: US20130244282A1
Автор: Ejebe Kenechi, Elbashir Sayda M., Schrum Jason P.
Принадлежит: Moderna Therapeutics

Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the use of modified messenger RNAs, and are useful for production of proteins. 1. A method of producing an antibody in a human cell , comprising contacting said human cell with a composition comprising:a. a first mRNA encoding the heavy chain of said antibody, andb. a second mRNA encoding the light chain of said antibody;wherein each of said first and said second mRNA contains at least one nucleoside modification and are each substantially devoid of uracil nucleotides.2. The method of claim 1 , wherein said at least one nucleoside modification is selected from the group consisting of pseudouridine.3. The method of claim 1 , wherein each of said first and second mRNA further contains the nucleoside modification 5-methyl-cytidine.4. The method of claim 1 , wherein the 5′ cap structure of each of said first and second mRNA is independently selected from the group consisting of Cap 1.5. The method of claim 1 , wherein the amount of antibody produced by said human cell is greater than the amount produced by a composition comprising said first and said second mRNA which are unmodified.6. The method of claim 1 , wherein in the composition the first mRNA and the second mRNA are in a 1:1 ratio. The present application is a continuation of U.S. patent application Ser. No. 13/252,049, filed Oct. 3, 2011, entitled Engineered Nucleic Acids and Methods of Use Thereof which claims the benefit of U.S. Provisional Application No. 61/404,413, filed Oct. 1, 2010, the disclosure of each of which is considered part of (and is incorporated herein by reference in) the disclosure of this application.The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled M008TR1SEQLST.tx created on May 17, 2013 which is 57,704 bytes in ...

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Номер записи: 46
19-09-2013 дата публикации

TARGETED PROBES OF CELLULAR PHYSIOLOGY

Номер: US20130244891A1
Автор: Bruchez Marcel P., Chakraborty Subhasish K., Schmidt Brigitte F., Waggoner Alan
Принадлежит: CARNEGIE MELLON UNIVERSITY

Biosensor comprising an activatable acceptor fluorogen linked via a linker to a donor which transfers energy to the fluorogen on detecting an analyte wherein the fluorogen component reacts and a 100 fold increase in intensity results when the fluorogen interacts non-covalently with an activator e.g. fluorogen activator peptide. 1. A biosensor comprising an activatable acceptor fluorogen linked by a linker to an environment-sensitive donor that interacts with an analyte , wherein the activatable acceptor fluorogen produces a fluorescence signal increase of at least 100-fold when it interacts non-covalently with an activator of the activatable acceptor fluorogen as compared to when no activator is present , and wherein the environment-sensitive donor transfers excitation energy to the activatable acceptor fluorogen such that , when activated , the activatable acceptor fluorogen produces a detectable fluorescent signal when the environment-sensitive donor is excited and the environment-sensitive donor transfers different amounts of excitation energy to the activatable acceptor fluorogen when it interacts with the analyte as compared to when no analyte is present.2. The biosensor of wherein the activator is a fluorogen activator peptide (FAP).3. The biosensor of claim 1 , wherein the activatable acceptor fluorogen and the environment-sensitive donor are covalently linked.4. The biosensor of claim 1 , wherein the environment-sensitive donor is attached to the activatable acceptor fluorogen by a molecular linker and the activatable acceptor fluorogen and the environment-sensitive donor are close enough for resonance energy transfer of excited state energy of the sensitive donor to be transferred to the activatable acceptor fluorogen with at least 50% efficiency.5. The biosensor of claim 1 , wherein the environment-sensitive donor detects an ion binding event leading to a change in the fluorescence intensity of the activatable acceptor fluorogen when it is bound to its ...

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Номер записи: 47
19-09-2013 дата публикации

REMOVAL OF PROTEIN AGGREGATES FROM BIOPHARMACEUTICAL PREPARATIONS IN A FLOW-THROUGH MODE

Номер: US20130245139A1
Автор: Cataldo William, Galipeau Kevin, Hamzik James, Kozlov Mikhail, Peeck Lars, Potty Ajish, Umana Joaquin
Принадлежит: EMD Millpore Corporation

The present invention provides novel compositions and methods for removal of protein aggregates from a sample in a flow-through mode. 1. A flow-through chromatography method of separating a monomeric protein of interest from protein aggregates in a sample , the method comprising contacting the sample with a solid support comprising one or more cation exchange binding groups attached thereto , at a density of about 1 to about 30 mM , wherein the solid support selectively binds protein aggregates , thereby to separate the monomeric protein of interest from protein aggregates.2. The method of claim 1 , wherein the solid support is selected from a chromatographic resin claim 1 , a membrane claim 1 , a porous bead claim 1 , a porous monolith claim 1 , a winged fiber claim 1 , a woven fabric and a non-woven fabric.3. The method of claim 1 , wherein the solid support is a porous polyvinylether polymeric bead or a porous crosslinked polymethacrylate polymer bead.4. The method of claim 1 , wherein the protein aggregates are lower order protein aggregates.5. The method of claim 4 , wherein the lower order protein aggregates are selected from the group consisting of dimers claim 4 , trimers claim 4 , and tetramers.6. The method of claim 1 , wherein the protein aggregates are high molecular weight protein aggregates.7. The method of claim 6 , wherein the higher molecular weight aggregates are pentamers and higher.8. The method of claim 1 , wherein the one or more cation exchange group is selected from the group consisting of a sulfonic group claim 1 , a sulfate group claim 1 , a phosphonic group claim 1 , a phosphoric group claim 1 , and a carboxylic group.9. The method of claim 1 , wherein the monomeric protein of interest is an antibody.10. The method of claim 9 , wherein the antibody is a monoclonal antibody.11. The method of claim 1 , wherein the monomeric protein of interest is a recombinant protein.12. The method of claim 1 , wherein the sample claim 1 , prior to ...

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Номер записи: 48
19-09-2013 дата публикации

Multispecific Molecules

Номер: US20130245233A1
Автор: Jing Li, Li Zhou, Ming Lei, Zhenping Zhu
Принадлежит: Individual

The present invention is directed to multi-specific/multivalent molecules of novel formats. The molecules with the formats of the present invention have desired yield and thermostability. The present invention also includes methods of producing and using the molecules described herein.

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Номер записи: 49
19-09-2013 дата публикации

Analysis of Ubiquitinated Polypeptides

Номер: US20130245237A1
Автор: Guo Ailan, II John Edward, LI Jing, Rush
Принадлежит: CELL SIGNALING TECHNOLOGY, INC.

The invention relates to antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis 117-. (canceled)18. An isolated antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant , wherein the antibody comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2 , SEQ ID NO: 3 , SEQ ID NO: 4 , SEQ ID NO: 5 , SEQ ID NO: 6 , SEQ ID NO: 7 , and SEQ ID NO: 8.1920-. (canceled)21. An isolated binding partner that specifically binds a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant.22. An isolated antibody that specifically binds a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant.23. The antibody of claim 22 , wherein the antibody is a monoclonal antibody.24. The antibody of claim 22 , wherein the antibody is a polyclonal antibody.25. The antibody of claim 22 , wherein the antibody is a human antibody.26. The antibody of claim 22 , wherein the antibody is a chimeric antibody.27. The antibody of claim 22 , wherein the antibody has a dissociation constant (kD) of 10M or less for the ubiquitin remnant peptide.28. The antibody of claim 22 , wherein the antibody has a dissociation constant (kD) of 10M or less for the ubiquitin remnant peptide.29. The antibody of claim 22 , wherein the antibody has an off rate (k) of 10/sec or less.30. The antibody of claim 22 , wherein the antibody has an off rate (k) of 10/sec ...

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Номер записи: 50
19-09-2013 дата публикации

Non-Human Animals Expressing pH-Sensitive Immunoglobulin Sequences

Номер: US20130247236A1
Автор: Andrew J. Murphy, Joel H. Martin, John Mcwhirter, Lynn Macdonald
Принадлежит: Regeneron Pharmaceuticals Inc

Genetically modified non-human animals are provided that express an immunoglobulin variable domain that comprises at least one histidine, wherein the at least one histidine is encoded by a substitution of a non-histidine codon in the germline of the animal with a hisidine codon, or the insertion of a histidine codon in a germline immunoglobulin nucleic acid sequence. Immunoglobulin genes comprising histidines in one or more CDRs, in an N-terminal region, and or in a loop 4 region are also provided. Immunoglobulin variable domains comprising one or more histidines (e.g., histidine clusters) substituted for non-antigen-binding non-histidine residues. Non-human animals that are progeny of animals comprising modified heavy chain variable loci (V, D, J segments), modified light chain variable loci (V, J segments), and rearranged germline light chain genes (VJ sequences) are also provided. Non-human animals that make immunoglobulin domains that bind antigens in a pH-sensitive manner are provided.

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Номер записи: 51
03-10-2013 дата публикации

DIMERIC MOLECULAR COMPLEXES WITH FREE CYSTEINE RESIDUES AND CONJUGATES THEREOF

Номер: US20130259806A1
Автор: Cornelius Aline, Light David, Seto Marian, Szymanski Paul
Принадлежит:

The invention relates generally to dimeric molecular complexes comprising two fusion proteins. Each fusion protein comprises a biological effector moiety, a polypeptide spacer sequence, and an IgE CH4 dimerization domain. The dimeric molecular complexes may be conjugated, at a defined site, to other molecules including drug moieties, cytotoxic agents, labels (such as detectable labels), or biocompatible polymers. 2. The molecular complex of claim 1 , wherein the biological effector moieties of the first and second fusion proteins each comprises an antigen binding site.3. (canceled)4. (canceled)5. The molecular complex of claim 1 , wherein i) cysteine residue in the biological effector moiety is replaced with a non-cysteine residue to create a free cysteine residue claim 1 , or ii) a non-cysteine residue in the CH4 dimerization domain is replaced with a cysteine residue to create a free cysteine residue.6. (canceled)7. The molecular complex of claim 1 , wherein i) the CH4 dimerization domain comprises the amino acid sequence of SEQ ID NO: 28 claim 1 , or ii) the CH4 dimerization domain comprises an amino acid sequence of SEQ ID NO: 28 that is mutated such that a non-cysteine residue at position Ala4 claim 1 , Val146 claim 1 , Ala51 claim 1 , Ala77 claim 1 , Lys82 claim 1 , Ser95 claim 1 , Gln101 claim 1 , Ala103 or Ser105 is replaced with a cysteine residue.8. (canceled)9. The molecular complex of claim 1 , wherein the first and the second fusion protein each further comprises an M2″ extension of an IgE splice variant located between the CH4 dimerization domain and the C-terminus of the protein.10. The molecular complex of claim 1 , wherein the polypeptide spacer sequences of both the first and second fusion protein each comprises an IgG hinge.11. (canceled)12. The molecular complex of claim 10 , wherein the hinge region of at least one of the first and the second fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29 ...

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Номер записи: 52
03-10-2013 дата публикации

Method for the production of immunoglobulin single variable domains

Номер: US20130261288A1
Автор: Ann Brige, Bart Walcarius, Mauro Sergi, Yves Meyvis
Принадлежит: Ablynx NV

The present disclosure relates to an improved method for the manufacture of immunoglobulin single variable domains. More specifically, the present disclosure relates to a method of producing immunoglobulin single variable domains in which the proportion of carbamylated variants is strongly reduced or absent and to improved immunoglobulin single variable domains obtainable by methods of the present disclosure.

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Номер записи: 53
10-10-2013 дата публикации

ANTIBODY-LINKED IMMUNO-SEDIMENTATION AGENT AND METHOD OF ISOLATING A TARGET FROM A SAMPLE USING SAME

Номер: US20130266930A1
Автор: Dinges Warren L.
Принадлежит: CytoSed, Inc.

The present disclosure is directed to antibody-linked immuno-sedimentation agent, the antibody being linked to a sedimentation agent by a non-antigen binding region of the antibody, and a method of isolating a target from a sample using the antibody-linked immuno-sedimentation agent. The methods involve forming a mixture including a sample with an antibody linked immuno-sedimentation agent and red blood cells under conditions sufficient to form red blood cell rouleaux and allow antibody-antigen binding. 1. A method of isolating a target from a sample , the method comprising: 1) at least one sedimentation agent and', '2) at least one antibody linked to the at least one sedimentation agent by a non-antigen binding region of the antibody;, 'a) forming a mixture comprising a sample, an antibody-linked immuno-sedimentation agent and red blood cells, wherein the antibody-linked immuno-sedimentation agent comprises'}b) incubating the mixture under conditions sufficient to form a rouleaux and allow the antibody to bind to an antigen present in the mixture; andc) recovering a target from the mixture.2. The method of claim 1 , wherein the rouleaux is formed by adsorption of the at least one sedimentation agent onto the red blood cells of the rouleaux.3. The method of claim 1 , wherein the at least one antibody is linked to the at least one sedimentation agent prior to forming the mixture.4. The method of claim 1 , wherein the antibody binds a target antigen or a non-target antigen in the sample.5. The method of claim 1 , further comprising incubating the mixture under conditions sufficient to form a rouleaux network.6. The method of claim 1 , wherein the sample is selected from the group consisting of whole blood claim 1 , blood plasma claim 1 , blood stem cell isolates claim 1 , platelet-rich plasma claim 1 , fractionated blood claim 1 , packed red blood cells claim 1 , umbilical cord blood claim 1 , bone marrow claim 1 , bone-marrow aspirates claim 1 , a buffy-coat layer ...

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Номер записи: 54
10-10-2013 дата публикации

METHOD FOR PREPARING AQUEOUS SOLUTION CONTAINING CULTURE MEDIUM AND CHELATING AGENT

Номер: US20130267684A1
Автор: Imamoto Yasufumi, Ishibashi Jun, Konno Yoshinobu, TAKAHASHI Ken, Tanaka Hisaya, Wakamatsu Kairo
Принадлежит: KYOWA HAKKO KIRIN CO., LTD.

Provided are a method for preparing a highly versatile aqueous solution having remarkably improved membrane filterability, which can be stably membrane-filtered in a short time, an aqueous solution prepared by the preparation method, a method for culturing cells using the aqueous solution which is prepared by the preparation method, a method for producing a physiologically active substance using the culturing method, a physiologically active substance produced by the method for producing a physiologically active substance, a method for performing membrane filtration of the aqueous solution which is prepared by the preparation method of the aqueous solution, a method for improving membrane filterability of the aqueous solution, and a method for producing the physiologically active substance by preparing the aqueous solution, performing membrane filtration of the aqueous solution, and then culturing cells using the resulting aqueous solution. The present invention relates to a method for preparing an aqueous solution, characterized by addition of a chelating agent. 1. A method for preparing an aqueous solution comprising a culture medium and a chelating agent , wherein the chelating agent is added to the aqueous solution prior to the final pH adjustment of the aqueous solution.2. The method for preparing an aqueous solution according to claim 1 , wherein the chelating agent is one or more selected from citric acid claim 1 , malic acid claim 1 , ethylenediaminetetraacetic acid claim 1 , ethylenediaminetetraacetic acid iron(III) sodium salt claim 1 , sialic acid claim 1 , and salts or hydrates thereof.3. The method for preparing an aqueous solution according to or claim 1 , wherein the culture medium is a powdered culture medium claim 1 , a liquid culture medium claim 1 , or a slurry culture medium.4. The method for preparing an aqueous solution according to claim 3 , wherein the powdered culture medium further includes one or more selected from metal salts claim 3 , ...

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Номер записи: 55
17-10-2013 дата публикации

Methods for processing inclusion bodies

Номер: US20130273607A1
Автор: Ellen T. O'Connor
Принадлежит: MEDIMMUNE LLC

The present application relates to methods for purifying recombinant proteins, including antibodies and antibody fragments. Suitably, the methods utilize depth filtration to clarify the desired proteins from a solubilized mixture, and provide refolding methods and refolding buffers to allow for refolding of the recombinant proteins into functional and active proteins. Exemplary antibody fragments include anti-CD22 antibody fragments that comprise V H and V L chains refolded into a functional and active fragment.

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Номер записи: 56
24-10-2013 дата публикации

CELL CULTURE COMPOSITIONS AND METHODS FOR POLYPEPTIDE PRODUCTION

Номер: US20130281355A1
Автор: AREVALO Silvana R., CARVALHAL Veronica, Gawlitzek Martin, MEIER Steven J., MUN Melissa S., VARMA Sharat, VIJAYASANKARAN Natarajan, Yang Yi, Zhang Boyan
Принадлежит:

Cell culture media, such as chemically defined cell culture media, are provided, as are methods of using the media for cell growth (i.e., cell culture) and polypeptide (e.g., antibody) production. Compositions comprising polypeptides produced by the methods are also provided. 1. A method of culturing cells , comprising the step of contacting the cells with a cell culture medium comprising:from about 300 mg/L to about 1200 mg/L cystine;from about 0.05 mg/L to about 1.0 mg/L vitamin B2;from about 0.05 mg/L to about 10.0 mg/L vitamin B6;from about 0.05 mg/L to about 12.0 mg/L vitamin B9; andfrom about 0.05 to about 2.5 mg/L vitamin B12.2. The method of claim 1 , comprising the step of contacting the cells with a cell culture medium comprising:from about 0.8 mM to about 2.5 mM cystine;from about 0.11 μM to about 0.72 μM vitamin B2;from about 4.5 μM to about 30.0 μM vitamin B6;from about 3.4 μM to about 22.0 μM vitamin B9; andfrom about 0.2 μM to about 1.5 μM vitamin B12.3. The method of claim 2 , wherein the cell culture medium further comprises any one or more of vitamin B 1 claim 2 , vitamin B3 claim 2 , vitamin B5 and vitamin B7.4. The method of claim 3 , wherein the cell culture medium further comprises any one or more of:from about 2.0 μM to about 14.0 μM vitamin B1;from about 11.0 μM to about 72.0 μM vitamin B3;from about 6.8 μM to about 44.0 μM vitamin B5; andfrom about 0.02 μM to about 0.14 μM vitamin B7.5. The method of claim 1 , wherein the cell culture medium further comprises an iron source.6. The method of claim 5 , wherein the iron source is ferric citrate or ferrous sulfate.7. The method of claim 1 , wherein cell culture medium comprises ferric citrate at a concentration of from about 2 μM to about 80 μM.8. The method of claim 7 , wherein the cell culture medium comprises ferric citrate at a concentration of from about 11.0 μM to about 36.0 μM.9. The method of claim 1 , wherein the cell culture medium further comprises hydrocortisone.10. The method of ...

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Номер записи: 57
24-10-2013 дата публикации

MODIFIED ANTIBODY WITH IMPROVED HALF-LIFE

Номер: US20130281677A1
Автор: Taura Tetsuya, Wilson David
Принадлежит: Cephalon Australia Pty, Ltd.

The present invention relates to antibodies, immunoglobulin constructs or immunoglobulin IgG4 fusion proteins whose in vivo half-lives are increased by the combination of (i) a modified IgG4 Fc region or FcRn binding domain thereof and (ii) a modified IgG4 hinge region sequence. 1. An isolated antibody , immunoglobulin construct or immunoglobulin IgG4 fusion protein with increased in vivo half-life , comprising:(i) a human IgG4 Fc region or the FcRn binding domain thereof modified relative to a corresponding unmodified IgG4 Fc region or FcRn binding domain thereof to comprise substitutions at one or more of amino acid residues 251-256 numbered according to the EU index as in Kabat; and(ii) a human IgG4 core hinge region sequence comprising a substitution of the serine residue within the amino acid sequence CPSCP (SEQ ID NO:1) to proline;wherein the in vivo half-life of the modified antibody, immunoglobulin construct or immunoglobulin IgG4 fusion protein is increased compared with the corresponding unmodified antibody, immunoglobulin construct or immunoglobulin IgG4 fusion protein.240-. (canceled) This document claims priority from U.S. Ser. No. 61/425,858, the entire contents of which are herein incorporated by reference.The present invention relates to antibodies, immunoglobulin constructs or immunoglobulin IgG4 fusion proteins whose in vivo half-lives are increased by the combination of (i) a modified IgG4 Fc region or FcRn binding domain thereof and (ii) a modified IgG4 hinge region sequence.IgG is the most prevalent immunoglobulin class in human and other mammals and is utilised in various types of immunotherapies and diagnostic procedures. One critical issue in these therapies is the period of persistence of immunoglobulins in the circulation. The rate of clearance of an administered immunoglobulin directly affects the amount and frequency of dosage of the immunoglobulin. Studies of IgG catabolism in the circulation have identified the portions of the IgG ...

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Номер записи: 58
14-11-2013 дата публикации

METHOD FOR USING LIGHT SCATTERING IN REAL TIME TO DIRECTLY MONITOR AND CONTROL IMPURITY REMOVAL IN PURIFICATION PROCESSES

Номер: US20130303732A1
Автор: Bailey Robert, HEWIG, III Arthur C., PHAN Duke H., YIGZAW Yinges
Принадлежит:

The invention provides a method for controlling contaminants in biopharmaceutical purification processes by using light scattering and UV absorbance to establish a determinant. The invention makes use of multi-angle light scattering (MALS) and UV as a continuous monitoring system to provide information about the elution peak fractions in real-time instead of conventional pooling methods that rely on a predetermined percent UV peak max value to initiate the pooling process; regardless of product quality. 1. A method for determining a stop collection point during ion exchange chromatography comprising:applying a mixture containing a recombinant protein and biological impurities on to an ion exchange chromatography resin;selectively eluting the recombinant protein from the chromatography resin;monitoring the eluate with a laser light scattering detector and an UV absorbance detector;obtaining the signals from the light scattering detector and the UV absorbance detector and calculating a fraction LS/UV ratio for each fraction until the peak max LS/UV is determined;calculating a normalized LS/UV ratio for all subsequent fractions, wherein an increase in the normalized LS/UV ratio indicates an increase in the amount of biological impurities in the eluate pool; andterminating the elution when the normalized LS/UV ratio reaches a predetermined value.2. The method according to claim 1 , wherein the laser light scattering detector is a single or multiple angle light scattering detector.3. The method according to claim 1 , wherein the light scattering detector and UV absorbance detector are in series or parallel.4. The method according to claim 1 , wherein the eluate is continuously delivered to the UV absorbance detector claim 1 , the laser light scattering detector claim 1 , or both5. The method according the claim 1 , wherein the entire eluate stream is delivered to the UV absorbance detector claim 1 , the laser light scattering detector or both.6. The method according the ...

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Номер записи: 59
21-11-2013 дата публикации

TARGETED THERAPEUTICS BASED ON ENGINEERED PROTEINS FOR TYROSINE KINASES RECEPTORS, INCLUDING IGF-IR

Номер: US20130310317A1
Автор: Camphausen Ray, FABRIZIO David, GAGE Patrick, MENDLEIN John, WRIGHT Martin C.
Принадлежит: BRISTOL-MYERS SQUIBB COMPANY

The present invention provides innovative proteins that bind to insulin-like growth factor-I receptor (IGF-IR), as well as other important proteins. The invention also provides innovative proteins in pharmaceutical preparations and derivatives of such proteins and the uses of same in diagnostic, research and therapeutic applications. The invention further provides cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and vectors comprising the polynucleotides encoding the innovative proteins. 1. A polypeptide comprising an altered tenth fibronectin type III (Fn3) domain , wherein the altered Fn3 domain (i) comprises an AB loop , BC loop , CD loop , DE loop , EF loop , and FG loop , wherein the amino acid sequences of the BC , DE , and FG loops of the Fn3 domain are at least 80% identical to the amino acid sequences of the respective BC , DE , and FG loops of an Fn3 domain with an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2-125 , 184-203 , or 226 , and (ii) binds human insulin-like growth factor-I receptor (IGF-IR) with a disassociation constant of about 1 μM or less.2. The polypeptide of claim 1 , wherein the amino acid sequences of the BC claim 1 , DE claim 1 , and FG loops of the Fn3 domain are identical to the amino acid sequences of the respective BC claim 1 , DE and FG loops of an Fn3 domain with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-125 claim 1 , 184-203 claim 1 , or 226.3. A polypeptide comprising an altered tenth fibronectin type III (Fn3) domain claim 1 , wherein the altered Fn3 domain (i) comprises an AB loop claim 1 , BC loop claim 1 , CD loop claim 1 , DE loop claim 1 , EF loop claim 1 , and FG loop claim 1 , wherein at least one loop of the BC claim 1 , DE claim 1 , and FG loops of the Fn3 domain has 1 claim 1 , 2 claim 1 , or 3 amino acid substitutions relative to the respective BC claim 1 , DE claim 1 , and FG loops of an Fn3 domain with an amino ...

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Номер записи: 60
28-11-2013 дата публикации

Culture method for obtaining a clonal population of antigen-specific b cells

Номер: US20130316353A1
Автор: Anne Elisabeth Carvalho-Jensen, Ethan Wayne Ojala, John A. Latham, Leon F. Garcia-Martinez
Принадлежит: Alder Biopharmaceuticals Inc

The present invention relates to methods of isolating antigen-specific cells and producing antibodies therefrom.

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Номер записи: 61
28-11-2013 дата публикации

Tandem Purification of Proteins

Номер: US20130317198A1
Автор: Coffman Jonathan Lee, Godavarti Ranganathan, Shamashkin Michael
Принадлежит: PFIZER INC.

The present disclosure provides methods for purifying products from a fluid. In some embodiments, provided purification methods use a combination of purification modes (e.g., protein A and ion exchange) operated in tandem, wherein at least one of the modes utilizes weak partitioning. In some embodiments, provided purification methods operate under robust conditions in which a degree of binding between a product and resin is maintained despite variations in operating parameters. 1. A method of recovering a purified protein product from a load fluid , the method comprising:a) exposing a load fluid comprising a protein product to a column comprising a first resin under conditions in which the product binds to the resin;b) recovering fluid comprising the product from the first resin to produce a first eluate;c) titrating the first eluate with a titrant as it passes to the second resin, wherein the titrant alters one or more conditions of the first eluate such when volumetric ratio of the eluate to the titrant varies by up to 40%, the change in partition coefficient of the product for the second resin is less than 10%;d) exposing the titrated eluate to a column comprising the second resin under conditions in which the product binds to the second resin, wherein the column comprising the first resin is arranged in tandem with the column comprising the second resin; ande) recovering fluid comprising the product from the second resin to produce a second eluate, thereby recovering a purified protein product from a load fluid.2. The method of claim 1 , wherein the first eluate is titrated with the titrant in a volumetric ratio of between about 95:5 to 80:20.3. The method of claim 1 , wherein a first pump delivers the load fluid to the first resin claim 1 , a second pump delivers the titrant to the first eluate claim 1 , and wherein the pumps are operated at a ratio of flow rates that varies by less than 30%.4. The method of claim 1 , wherein the titrant and first eluate are ...

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Номер записи: 62
28-11-2013 дата публикации

Methods of Purifying Antibodies

Номер: US20130317200A1
Автор: Depoisier Jean-Francois, Elson Greg, Fischer Nicolas, Fouque Nicolas, Magistrelli Giovanni
Принадлежит: Novlmmune S.A.

The invention provides methods of purifying antibodies using various antibody-specific purification media to rapidly and efficiently separate mixtures of antibodies, antibody fragments and/or antibody components to isolate a desired antibody product from the mixture. The invention relates to the purification of bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, e.g., antibodies composed of a single heavy chain and two different light chains, one containing a Kappa constant domain and the other a Lambda constant domain, including antibodies of different specificities that share a common heavy chain. The invention also provides the methods of efficiently purifying intact antibodies by separating the intact antibody from non-intact antibodies including free light chains. 1. A method of purifying a bispecific monoclonal antibody carrying a different specificity in each combining site and consisting of two copies of a single heavy chain polypeptide and a first light chain comprising a kappa constant region and a second light chain comprising a lambda constant region , the method comprising the steps of:(a) providing a mixed antibody composition that comprises one or more of the bispecific monoclonal antibodies carrying a different specificity in each combining site and consisting of two copies of a single heavy chain polypeptide and a first light chain comprising a kappa constant region and a second light chain comprising a lambda constant region (bispecific MAb); one or more monospecific monoclonal antibodies having two lambda light chains or portions thereof (λ-MAb); and one or more monospecific monoclonal antibodies having two kappa light chains or portions thereof (κ-MAb);(b) providing a separation means that has a specific affinity for a kappa light chain constant region or a lambda light chain constant region;(c) contacting the separation means with the mixed antibody composition under conditions ...

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Номер записи: 63
05-12-2013 дата публикации

REPEAT-CHAIN FOR THE PRODUCTION OF DIMER, MULTIMER, MULTIMER COMPLEX AND SUPER-COMPLEX

Номер: US20130323717A1
Автор: Choe MuHyeon, Lee YongChan, Won JaeSeon
Принадлежит:

The present invention relates to a method for manufacturing multimers by making repeat-chains comprising repeatedly linked affinity domains binding specifically to monomers, and by using the same to create a repeat-chain/multiple-monomer complex created from the repeat-chains and a multiple number of monomers, thereby facilitating the formation of bond bridges between the monomers in the complex to produce inter-monomeric bond bridged multimer. 115.-. (canceled)16. A method for preparing a super-complex , which comprises the following steps:1) preparing repeat-chains which contain a single or multiple kinds of monomer-specific binding domains having at least two binding sites for a monomer repeated therein;2) preparing repeat-chain/multiple-monomer complexes by mixing the repeat-chains of step 1) and the monomers having at least two binding sites for the repeat-chains; and3) generating sugar-complex aggregates of the complexes by forming cross-binding between the repeat-chain/multiple-monomer complexes of step 2).17. The method as set forth in claim 16 , wherein the monomer is a protein.18. The method as set forth in claim 16 , wherein the monomer is selected from the group consisting of antibodies claim 16 , ligands claim 16 , receptors or fragments thereof claim 16 , or recombinants thereof claim 16 , or derivatives thereof claim 16 , or fusions of biological or chemical functional group therewith.19. The method as set forth in claim 18 , wherein the antibody is selected from the group consisting of fragments of an antibody claim 18 , Fab fragment claim 18 , fragments containing Fab fragment claim 18 , Fv fragment claim 18 , fragments containing Fv fragment claim 18 , Fc fragment claim 18 , and fragments containing Fc fragment.20. The method as set forth in claim 16 , wherein the binding domain is a protein.21. (canceled)22Staphylococcus aureusPeptostreptococcus magnus. The method as set forth in claim 16 , wherein the binding domain is selected from the group ...

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Номер записи: 64
12-12-2013 дата публикации

METHODS FOR CULTURING HUMAN MYELOID LEUKAEMIA CELLS AND CELLS DERIVED THEREFROM

Номер: US20130330768A1
Автор: Stahn Rainer
Принадлежит: GLYCOTOPE GmbH

The present invention pertains to a method for culturing a suspension of immortalized human blood cells, preferably cells of myeloid leukaemia origin or cells derived therefrom, wherein said method provides a high productivity, a high cell viability and growth rate and a high batch-to-batch consistency, and can be scaled up without altering these parameters. 1. A method for culturing a suspension of immortalized human blood cells in cell culture medium , wherein said method has one or more of the following characteristics:a) said suspension is agitated such that the resulting specific power input is at least 0.005 W/kg,b) said suspension is agitated with an intensity that is suitable for allowing an exclusive flow gas supply of said suspension,{'sup': '2', 'c) said suspension is agitated such that the resulting shear force is at least 0.1N/m,'}{'sup': '−1', 'd) said suspension is agitated such that the resulting shear rate at the tip of the stirrer is at least 300 s, if a stirrer is used for agitation,'}e) said cells are supplied with at least one gas by exclusive flow,f) said cells are supplied with at least one gas at a flow rate of at most 0.05 l/h per litre of reactor volume,g) the gas supply has a peak flow rate of 0.05 vvm or less.2. The method of claim 1 , wherein the specific power input for agitation of the suspension is at least 0.01 W/kg and the gas supply is achieved by exclusive gas flow3. The method according to claim 2 , wherein the peak gas flow is 0.05 vvm or less.4. A method for the recombinant production of a product of interest in immortalized human blood cells claim 1 , wherein said cells comprise a gene encoding the product of interest and wherein said cells are cultured according to the method of .5. The method according to claim 4 , wherein the expressed product of interest is obtained by purifying the product of interest from the cell culture medium.6. The method of claim 1 , wherein the immortalized human blood cells are human myeloid ...

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Номер записи: 65
12-12-2013 дата публикации

MEANS AND METHODS FOR PRODUCING HIGH AFFINITY ANTIBODIES

Номер: US20130331552A1
Автор: Bakker Adrianus Quirinus, Beaumont Tim, Kwakkenbos Mark Jeroen, Spits Hergen, Wagner Koen
Принадлежит: AIMM Therapeutics B.V.

Described are means and methods for producing high-affinity antibodies against an antigen of interest, using stable B-cell cultures. 1. A method for producing antibodies specific for an antigen of interest , the method comprising the steps of:a) selecting a B-cell able to produce antibody specific for said antigen of interest or selecting a B-cell able to differentiate into a B-cell that is able to produce antibody specific for said antigen of interest;b) inducing, enhancing and/or maintaining expression of BCL6 in said B-cell;c) inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid in said B-cell;d) allowing expansion of said B-cell into a population of said B-cells;e) selecting at least one B-cell from said population of B-cells producing a B-cell receptor and/or antibody with a binding capacity higher than the average binding capacity of said population of B-cells for said antigen of interest;f) culturing said at least one B-cell into a population of B-cells; andg) obtaining antibodies produced by the B-cell culture.2. The method according to claim 1 , further comprising claim 1 , following step e) claim 1 , repeating steps d) and e) at least once.3. The method according to claim 1 , wherein said at least one B-cell is cultured for at least four weeks.4. The method according to claim 1 , wherein said B-cell selected in step a) is a memory B-cell.5. The method according to claim 1 , wherein said anti-apoptotic nucleic acid comprises a gene encoding an anti-apoptotic molecule.6. The method according to claim 1 , further comprising:providing said B-cell with a growth factor.7. The method according to claim 1 , further comprising:directly or indirectly controlling the amount of BLIMP1 expression product in said B-cell selected in step a).8. The method according to claim 1 , wherein said B-cell selected in step a) originates from an individual that had been previously exposed to said antigen of interest.9. The method according to claim 1 ...

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Номер записи: 66
19-12-2013 дата публикации

NOVEL PURIFICATION OF HUMAN, HUMANIZED, OR CHIMERIC ANTIBODIES USING PROTEIN A AFFINITY CHROMATOGRAPHY

Номер: US20130336957A1
Автор: Huelsman Randolf, Lacy Susan, Wang Chen
Принадлежит: ABBVIE, INC.

Disclosed herein are compositions and methods for the isolation and purification of antibodies from a sample matrix. In particular, the present invention relates to compositions and methods for isolating and purifying antibodies exhibiting low or high binding capacity for Protein A resin. In certain embodiments, the methods herein employ a kosmotropic salt solution, an affinity chromatographic step, and may include one or more additional chromatography and/or filtration steps to achieve the desired degree of purification. The present invention is also directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein. 2. The method of claim 1 , wherein said antibody has weak binding strength and low binding capacity for the affinity chromatography resin.3. The method of claim 1 , wherein said antibody is a human antibody claim 1 , humanized antibody claim 1 , a chimeric antibody claim 1 , a multivalent antibody claim 1 , a dual-variable domain antibody claim 1 , or an antigen-binding portion thereof.4. The method of claim 1 , wherein said antibody in said primary recovery sample that is contacted to said affinity chromatography resin is concentrated such that it has a concentration of from about 1 g/L to about 10 g/L.5. The method of claim 1 , wherein the kosmotropic salt in said kosmotropic salt solution is selected from the group consisting of ammonium sulfate claim 1 , sodium sulfate claim 1 , sodium citrate claim 1 , potassium sulfate claim 1 , potassium phosphate claim 1 , sodium phosphate claim 1 , and a combination thereof.6. The method of claim 5 , wherein said kosmotropic salt is present in said kosmotropic salt solution at a concentration of from about 0.3 M to about 1.1 M.7. The method of claim 1 , wherein said Protein A affinity chromatography resin is selected from any commercial Protein A resins including MabSelect SuRe™ claim 1 , MabSelect claim 1 , MabSelect SuRe LX claim 1 , MabSelect Xtra claim 1 , ...

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Номер записи: 67
09-01-2014 дата публикации

METHOD FOR INACTIVATION/REMOVAL OF COAGULATION FACTORS BY PRECIPITATION

Номер: US20140007547A1
Автор: Ahrer Karin, Kaar Waltraud, Zochling Alfred
Принадлежит: Octapharma AG

A method for inactivation or removal of coagulation factors FII, FVII, FVIIa, FIX, FIXa, FX, FXI and FXIa in or from protein containing solutions obtained from blood, blood plasma, plasma fractions or by recombinant means wherein the protein containing solution is contacted with an organic acid or its salt while being stirred. 1. A method for inactivation or removal of coagulation factors FII , FVII , FVIIa , FIX , FIXa , FX , FXI and/or FXIa in or from protein containing solutions obtained from blood , blood plasma , plasma fractions or by recombinant means wherein the protein containing solution is contacted with an organic acid or its salt while being stirred.2. The method of wherein the organic acid is a Cto C claim 1 , saturated or unsaturated organic acid or its salts.3. The method of wherein the organic acid is selected from butyric (C4) claim 1 , valeric (C5) claim 1 , caproic (C6) claim 1 , enanthic (C7) claim 1 , caprylic (C8) claim 1 , pelargonic (C9) and capric (C10) acid or its salts.4. The method of wherein the organic acid is caprylic acid or capric acid or a salt of these acids thereof.5. The method of wherein the method comprises contacting of the solution with an organic acid or its salt at a temperature of about 1 to about 40° C.6. The method of wherein the method comprises a first contacting of the solution with an organic acid or its salt at a temperature of about 2 to about 8° C. and a second contacting with the same acid or its salt or a different acid or its salt at a temperature of about 20 to about 30° C.7. The method of wherein the contacting takes place within a pH range of from 4.0 to 6.0.8. The method of wherein the contacting takes place within a pH range of from 4.2 to 5.6.9. The method of wherein the organic acid or its salt is added to the protein containing solution to a concentration of from 5 to 50 mmol/l.10. The method of wherein the organic acid or its salt is added to the protein containing solution to a concentration of from ...

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Номер записи: 68
09-01-2014 дата публикации

BI- AND MONOSPECIFIC, ASYMMETRIC ANTIBODIES AND METHODS OF GENERATING THE SAME

Номер: US20140010814A1
Автор: Benhar Itai, VAKS Lilach
Принадлежит: Ramot at Tel-Aviv University Ltd.

An antibody is provided. The antibody comprises an Fc region and a Fab region, wherein: 1. A bispecific antibody comprising an Fc region and a Fab region , wherein:(i) said Fc region comprises two non-identical heavy chains, wherein at least one of said two non-identical heavy chains comprises an amino acid modification so as to form a steric complementation between said two non-identical heavy chains, wherein said Fc region comprises a protuberance of one heavy chain of said Fc region and a sterically compensatory cavity on a second heavy chain of said Fc region, said protuberance protruding into said compensatory cavity, wherein said complementation is generated by:(a) substituting an amino acid at one position on a CH3 domain of said one heavy chain with another amino acid having a larger side chain volume than the original amino acid, said amino acid having a larger side chain being selected from the group consisting of tyrosine, arginine, phenylalanine, isoleucine and tryptophan so as to generate said protuberance; and(b) substituting an amino acid at one position on a CH3 domain of said second heavy chain with another amino acid having a smaller side chain volume than the original amino acid so as to generate said compensatory cavity, said amino acid having a smaller side chain being selected from the group consisting of alanine, glycine, valine and threonine; and{'sub': H', 'L, '(ii) said Fab region comprises a first covalent link between a first heavy chain and a first light chain of said Fab region and a second covalent link between a second heavy chain and a second light chain of said Fab region, wherein said first covalent link is a naturally occurring disulfide bond between a CH1 domain of said one heavy chain and a CL domain of said one light chain; and said second covalent link is a disulfide bond between a cysteine at position 44 of a Vdomain of said second heavy chain and a cysteine at position 100 of a Vdomain of said second light chain, wherein the ...

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Номер записи: 69
09-01-2014 дата публикации

Novel purification of non-human antibodies using protein a affinity chromatography

Номер: US20140010820A1
Автор: Chen Wang, Randolf Huelsman, Susan Lacy
Принадлежит: AbbVie Inc

Disclosed herein are compositions and methods for the isolation and purification of antibodies from a sample matrix. In particular, the present invention relates to compositions and methods for isolating and purifying antibodies exhibiting weak binding strength and low binding capacity for Protein A resin. In certain embodiments, the methods herein employ a kosmotropic salt solution, an affinity chromatographic step, and may include one or more additional chromatography and/or filtration steps to achieve the desired degree of purification. The present invention is also directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein.

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Номер записи: 70
09-01-2014 дата публикации

Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same

Номер: US20140013456A1
Автор: MACDONALD Lynn, MCWHIRTER John, MURPHY Andrew J.
Принадлежит: Regeneron Pharmaceuticals, Inc.

A genetically modified non-human animal is provided, wherein the non-human animal expresses an antibody repertoire capable of pH dependent binding to antigens upon immunization. A genetically modified non-human animal is provided that expresses human immunoglobulin light chain variable domains derived from a limited repertoire of human immunoglobulin light chain variable gene segments that comprise histidine modifications in their germline sequence. Methods of making non-human animals that express antibodies comprising histidine residues encoded by histidine codons introduced into immunoglobulin light chain nucleotide sequences are provided. 1. A genetically modified non-human animal comprising in its germline an immunoglobulin light chain locus comprising at least one human Vgene segment and at least one human Jgene segment operably linked to an immunoglobulin light chain constant region sequence ,{'sub': L', 'L, 'wherein each human Vgene segment comprises at least one histidine codon that is not encoded by the corresponding human germline Vgene segment, and'}{'sub': L', 'L, 'wherein the at least one human Vgene segment and the at least one human Jgene segment are capable of rearranging and encoding a human light chain variable domain of an antibody.'}2. The animal of claim 1 , wherein the animal does not comprise an endogenous Vgene segment that is capable of rearranging to form an immunoglobulin light chain.3. The animal of claim 1 , wherein the immunoglobulin light chain constant region sequence is a non-human immunoglobulin light chain constant region sequence.4. The animal of claim 3 , wherein the non-human immunoglobulin light chain constant region sequence is a mouse or a rat sequence.5. The animal of claim 3 , wherein the non-human immunoglobulin light chain constant region sequence is an endogenous immunoglobulin light chain constant region sequence.6. The animal of claim 1 , further comprising in its germline an immunoglobulin heavy chain locus that ...

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Номер записи: 71
16-01-2014 дата публикации

Methods of Modifying Eukaryotic Cells

Номер: US20140017229A1
Автор: Karow Margaret, MACDONALD Lynn, MURPHY Andrew J., STEVENS Sean, YANCOPOULOS George D.
Принадлежит: Regeneron Pharmaceuticals, Inc.

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification. 1. A method of producing an antibody having a human variable region , the method comprising exposing a genetically modified rodent to an antigen , the rodent comprising in its germline human unrearranged variable region gene segments inserted at an endogenous rodent immunoglobulin locus such that the rodent produces an antibody to the antigen having a human variable region.2. The method of claim 1 , wherein the human unrearranged variable region gene segments are heavy chain gene segments claim 1 , and the rodent immunoglobulin locus is a heavy chain locus.3. The method of claim 1 , wherein the human unrearranged variable region gene segments are light chain gene segments and the rodent immunoglobulin locus is a light chain locus.4. The method of claim 3 , wherein the light chain gene segments are human kappa light chain gene segments.5. The method of claim 1 , wherein the unrearranged variable gene segments are contained on a genomic DNA fragment larger than 20 kb.6. The method of claim 1 , wherein the human variable region of the antibody is encoded by the variable region gene segments following rearrangement.7. The method of claim 1 , wherein the antibody has a human variable region and a rodent constant region.8. The method of claim 1 , wherein the rodent does not ...

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Номер записи: 72
16-01-2014 дата публикации

Methods of Modifying Eukaryotic Cells

Номер: US20140017238A1
Автор: Karow Margaret, MACDONALD Lynn, MURPHY Andrew J., STEVENS Sean, YANCOPOULOS George D.
Принадлежит: Regeneron Pharmaceuticals, Inc.

A method for engineering and utilizing large DNA vectors to target, via homologous recombination, and modify, in any desirable fashion, endogenous genes and chromosomal loci in eukaryotic cells. These large DNA targeting vectors for eukaryotic cells, termed LTVECs, are derived from fragments of cloned genomic DNA larger than those typically used by other approaches intended to perform homologous targeting in eukaryotic cells. Also provided is a rapid and convenient method of detecting eukaryotic cells in which the LTVEC has correctly targeted and modified the desired endogenous gene(s) or chromosomal locus (loci) as well as the use of these cells to generate organisms bearing the genetic modification. 1. A method of producing an antibody having a human variable region , the method comprising exposing a genetically modified mouse to an antigen , the mouse comprising in its germline human unrearranged variable region gene segments inserted at an endogenous mouse immunoglobulin locus such that the mouse produces an antibody to the antigen having a human variable region.2. The method of claim 1 , wherein the human unrearranged variable region gene segments are heavy chain gene segments claim 1 , and the mouse immunoglobulin locus is a heavy chain locus.3. The method of claim 1 , wherein the human unrearranged variable region gene segments are light chain gene segments and the mouse immunoglobulin locus is a light chain locus.4. The method of claim 3 , wherein the light chain gene segments are human kappa light chain gene segments.5. The method of claim 1 , wherein the unrearranged variable gene segments are contained on a genomic DNA fragment larger than 20 kb.6. The method of claim 1 , wherein the human variable region of the antibody is encoded by the variable region gene segments following rearrangement.7. The method of claim 1 , wherein the antibody has a human variable region and a mouse constant region.8. The method of claim 1 , wherein the mouse does not comprise ...

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Номер записи: 73
16-01-2014 дата публикации

FOCUSED LIBRARIES OF GENETIC PACKAGES

Номер: US20140018261A9
Автор: Ladner Robert Charles
Принадлежит: DYAX CORP.

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies. 143.-. (canceled)44. A library , comprising a first set of variegated DNA molecules encoding a collection of antibody heavy chains (HC) , wherein each comprises a CDR1 region , a CDR2 region , and a CDR3 region , and wherein at least a heavy chain portion of the collection comprises a plurality of components that contain HC CDR3 regions , the components being selected from the group consisting of:(i) YYCA21111YFDYWG (SEQ ID NO:6), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;(ii) YYCA2111111YFDYWG (SEQ ID NO:7), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;(iii) YYCA211111111YFDAYTG (SEQ ID NO:8), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;(iv) YYCAR111S2S3111YFDYWG (SEQ ID NO:9), wherein 1 is any amino acid residue except C, 2 is a mixture of S and G, and 3 is a mixture of Y and W;(v) YYCA2111CSG11CY1YFDYWG (SEQ ID NO:10), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;(vi) YYCA211S1TIFG11111YFDYWG (SEQ ID NO:11), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;(vii) YYCAR111YY2S3344111YFDYWG (SEQ ID NO:12), wherein 1 is any amino acid residue except C, 2 is D or S, and 3 is a mixture of S and G;(viii) YYCAR1111YC2231CY111YFDYWG (SEQ ID NO:13), wherein 1 is any amino acid residue except C, 2 is a mixture of S and G, and 3 is a mixture of T, D and G; and(ix) a mixture of any of (i) to (viii) set forth above.45. The library of claim 44 , wherein:{'sup': '5', 'the complexity of component (i) is 2×10,'}{'sup': '7', 'the complexity of ...

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Номер записи: 74
16-01-2014 дата публикации

Methods for the identification and repair of amino acid residues destabilizing single-chain variable fragments (scFv)

Номер: US20140018521A1
Автор: Buchholz Christian, Schneider Irene
Принадлежит: Paul-Ehrlich-Institute of the Federal Republic of Germany

The invention relates to a method for identifying stabilizing amino acids in single chain antibody fragments. 1. A method of optimizing the stability of a single-chain variable fragment (scFv) , the scFv comprising an immunoglobulin heavy chain variable region (V) and an immunoglobulin light chain kappa variable region (V) , the method comprising the steps:{'sub': H', 'Lκ, 'a) identifying the amino acids located at positions 77 and 97 of the Vand the amino acids located at positions 44 and 105 of the Vof the scFv, wherein the positions are designated as published by Honegger and Plückthun (JMB (2001) 309:957-670); and'}{'sub': H', 'H', 'Lκ', 'Lκ, 'b) replacing the amino acid at position 77 of the Vby K, replacing the amino acid at position 97 of the Vby T, replacing the amino acid at position 44 of the Vby Y and replacing the amino acid at position 105 of the Vby Y, or'}{'sub': H', 'H', 'Lκ', 'Lκ, 'replacing the amino acid at position 77 of the Vby K, replacing the amino acid at position 97 of the Vby T, replacing the amino acid at position 44 of the Vby F and replacing the amino acid at position 105 of the Vby F.'}2. A method for evaluating the stability of a single-chain variable fragment (scFv) , comprising the steps:a) expressing in a mammalian cell line a fusion protein comprising N-terminally a first amino acid sequence comprising a mammalian cellular export signal, and C-terminally a second amino acid sequence comprising the amino acid sequence of an scFv;b) determining whether the fusion protein is exported from the inside of the cell to the outside of the cell; andc) designating scFvs comprised in fusion proteins that are exported from the inside of the cell to the outside of the cell as stable, and designating scFvs comprised in fusion proteins that are not exported from the inside of the cell to the outside of the cell as unstable.3. The method according to for optimizing the stability of an scFv claim 2 , further comprising the steps:d) analyzing the ...

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Номер записи: 75
23-01-2014 дата публикации

Activatable Antibodies Having Non-Binding Steric Moieties and Methods of Using the Same

Номер: US20140023664A1
Автор: Henry Bernard Lowman, Shouchun Liu
Принадлежит: Cytomx Therapeutics Inc

The invention relates generally to activatable antibodies and methods of making and using these activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

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Номер записи: 76
23-01-2014 дата публикации

KEX2 Cleavage regions of recombinant fusion proteins

Номер: US20140024067A1
Автор: Wang Huaming, Ward Michael
Принадлежит: DANISCO US INC.

The invention relates to a fusion DNA construct comprising a KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site, a fusion polypeptide, vectors and cells comprising the fusion DNA construct, methods for producing desired proteins from filamentous fungal cells and methods for enhancing the secretion and/or cleavage of a desired protein from a cell. 130-. (canceled)32. The method according to claim 31 , wherein the desired protein is a therapeutic protein.33. The method according to claim 31 , wherein the desired protein is an antibody.34. The method according to claim 31 , wherein the cleavage of the desired protein is increased compared to the cleavage of said desired protein from an equivalent fusion polypeptide lacking the KEX2 site pre-sequence.3526. The method according to claim claim 31 , wherein the KEX2 region is XXXXKR and Xis V and Xis E claim 31 , S claim 31 , T or Y.36. A method for increasing the production of an antibody from a filamentous fungal cell comprising: i) a promoter,', 'ii) a first DNA molecule encoding a signal sequence,', 'iii) a second DNA molecule encoding a carrier protein,', {'sub': 4', '3', '2', '1, 'claim-text': [{'sub': '4', 'Xis V, S, N, L, or K;'}, {'sub': '3', 'Xis A, V, D, W, E or P;'}, {'sub': '2', 'Xis V, I, L or F; and'}, {'sub': '1', 'Xis E, S, T or Y, and'}], 'iv) a third DNA molecule encoding a KEX2 region XXXXKR, wherein'}, 'v) a fourth DNA molecule encoding a desired protein;, 'a) obtaining a filamentous fungal cell comprising a fusion DNA construct encoding a fusion polypeptide comprising in operable linkage from the 5′ end of said constructb) culturing the fungal cell under suitable conditions for expression of the fusion polypeptide; andc) wallowing secretion of the fusion polypeptide,wherein the secretion of the desired protein is increased compared to the secretion of an equivalent fusion polypeptide not including a KEX2 pre-sequence.37. The method of claim 36 , wherein the ...

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Номер записи: 77
23-01-2014 дата публикации

METHODS FOR PRODUCING BIOLOGICAL MATERIALS

Номер: US20140024079A1
Автор: Yun Anthony Joonkyoo
Принадлежит:

Methods of producing biological materials from cells and organisms are provided. Aspects of the methods include modulating the stress conditions of the cells and/or organism to produce biological materials having one or more desired properties. In certain aspects, the cell or organism is evaluated to detect the presence or absence of a stressed phenotype, wherein an unstressed phenotype may be produced before the cell or organism produces the biological material of interest. The biological materials produced from such cells and organisms may be used for a variety of applications, including therapeutic, research, and other applications. 1. A method for producing a biological product from a cell , the method comprising:culturing the cell containing nucleic acid encoding at least a portion of the biological product, under conditions allowing for expression of the biological product, and wherein the conditions modulate a stressed phenotype of the cell; andrecovering the biological product.2. The method according claim 1 , comprising evaluating the cell to identify the presence or absence of a stressed phenotype.3. The method according to claim 2 , wherein evaluating the cell to identify the presence or absence of a stressed phenotype is performed prior to recovering the biological product.4. The method according claim 1 , comprising producing an unstressed phenotype in the cell.5. The method according to claim 4 , wherein producing an unstressed phenotype comprises changing the cell's nutritional intake.6. The method according to claim 4 , wherein producing an unstressed phenotype comprises changing the cell's environment.7. The method according to claim 6 , wherein the unstressed phenotype is produced by changing the cell's temperature or the cell's exposure to light.8. The method according to claim 4 , comprising administering to the cell an agent to reduce stress.9. (canceled)10. The method according to claim 4 , wherein the evaluating comprises visually inspecting ...

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Номер записи: 78
23-01-2014 дата публикации

HOST CELL FOR MAKING ANTIBODY FC-HETERODIMERIC MOLECULES USING ELECTROSTATIC STEERING EFFECTS

Номер: US20140024111A1
Автор: Kannan Gunasekaran, Pentony Martin J., Wittekind Michael, Yan Wei
Принадлежит: Amgen Inc.

The invention relates to methods of making Fc-heterodimeric proteins or polypeptides. The invention also relates to the Fc-heterodimeric proteins or polypeptides themselves, including the individual polypeptide components that comprise the heterodimer Nucleic acids encoding such polypeptides, expression vectors, and host cells. Moreover, the invention relates to pharmaceutical compositions comprising one of more Fc-heterodimeric proteins or polypeptides. 1. A host cell for producing a heterodimeric protein , said host cell comprising a nucleic acid encoding a first CH3-containing polypeptide and a nucleic acid encoding a second CH3-containing polypeptide , wherein said first human CH3-containing polypeptide comprises a replacement of the amino acid at position 392 with a negative-charged amino acid and said second human IgG CH3-containing polypeptide comprises a replacement of Asp399 , Glu356 , Asp356 , or Glu357 with a positive-charged amino acid.2. The host cell of claim 1 , wherein Lys392 is replaced with a negative-charged amino acid.3. The host cell of claim 1 , wherein Asn392 is replaced with a negative-charged amino acid.4. The host cell of claim 1 , wherein said first human CH3-containing polypeptide further comprises Lys409 or Arg409 replaced with a negative-charged amino acid.5. The host cell of claim 1 , wherein Lys392 or Asn392 is replaced with aspartic acid.6. The host cell of claim 4 , wherein said Lys409 or Arg409 is replaced with aspartic acid.7. The host cell of claim 1 , wherein said second human IgG CH3-containing polypeptide comprises a replacement of Asp399 claim 1 , Glu356 claim 1 , Åsp356 claim 1 , or Glu357 with lysine.8. The host cell of claim 1 , wherein said second human IgG CH3-containing polypeptide comprises a replacement of Asp399 and Glu356 with lysine.9. The host cell of claim 1 , wherein the heterodimeric protein comprises a human IgG Fc region.10. The host cell of claim 9 , wherein the human IgG Fc region comprises an IgG1 Fc ...

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Номер записи: 79
30-01-2014 дата публикации

Recombinant polypeptide production method

Номер: US20140030758A1
Автор: Hisahiro Tabuchi, Tomoya Sugiyama
Принадлежит: Chugai Pharmaceutical Co Ltd

Provided is a method capable of producing a protein at a high level using a cultured animal cell, comprising culturing a cell that expresses APES (Antibody Production Enhancing Sequence) and into which a DNA encoding a desired polypeptide has been introduced, thereby producing the desired polypeptide. APES contains a nucleotide sequence related to NfkBia and has a function of decreasing the intracellular expression of NfkBia.

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Номер записи: 80
30-01-2014 дата публикации

METHOD OF CLONING NUCLEIC ACID

Номер: US20140030761A1
Автор: Chen Chao-Guang, Panousis Con
Принадлежит: CSL LIMITED

The present disclosure provides methods for producing expression constructs comprising linking a plurality of unlinked nucleic acids, including nucleic acid encoding a marker protein. 1. A method for producing an expression construct capable of expressing a polypeptide , the method comprising linking the following unlinked nucleic acids:(i) a nucleic acid encoding the polypeptide; and(ii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein; and(iii) an expression vector, which does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (ii),such that the nucleic acid encoding the marker protein is positioned between the nucleic acid encoding the polypeptide and the expression vector.2. A method for producing an expression construct capable of expressing a plurality of polypeptides , the method comprising linking the following unlinked nucleic acids:(i) a nucleic acid encoding a first polypeptide;(ii) a nucleic acid encoding a second polypeptide; and(iii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein; and(iv) an expression vector, which does not comprise a nucleic acid encoding the marker protein encoded by the nucleic acid at (iii).3. A method for producing an expression construct capable of expressing a plurality of polypeptides comprising at least a first polypeptide and a second polypeptide , the method comprising linking the following nucleic acids:(i) a nucleic acid encoding the first polypeptide;(ii) a nucleic acid encoding a marker protein that facilitates detection and/or isolation of a cells expressing the marker protein; and(iii) a nucleic acid encoding the second polypeptide,such that the nucleic acid encoding the marker protein is positioned between the first and second nucleic acids.4. The method of claim 2 , wherein the first and second polypeptides associate to form a ...

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Номер записи: 81
06-02-2014 дата публикации

CELL SURFACE DISPLAY USING PDZ DOMAINS

Номер: US20140038842A1
Автор: Huang Chao B., Rondon Isaac J., Tam Eric M., Votin Violet
Принадлежит: XOMA TECHNOLOGY

Novel materials and methods useful for displaying polypeptides on the surface of a cell are provided, including cell surface proteins fused to a PDZ domain peptide and antibodies fused to PDZ-binding peptides. 1. A host cell comprising:(a) a polynucleotide encoding a cell surface protein fused to a PDZ Domain; and(b) a polynucleotide encoding an antibody, or antigen-binding fragment thereof, fused to a PDZ-binding peptide.2. The host cell of claim 1 , where the PDZ-binding peptide is 5 to 20 amino acids in length.3. The host cell of claim 1 , wherein the cell is selected from the group consisting of a eukaryotic cell and a prokaryotic cell.4. The host cell of wherein the eukaryotic cell is a yeast cell or a mammalian cell.5S. cerevisiae, P. pastoris, C. albicans, H. polymorpha, Y. lipoliticaS. pombe.. The host cell of wherein the yeast cell is selected from the group consisting of claim 4 , and6E. coli, Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosaSerratia marcescans.. The host cell of wherein the prokaryotic cell is selected from the group consisting of claim 3 , and7. The host cell of claim 1 , wherein the cell surface protein is a cell wall protein.8. The host cell of claim 7 , wherein the cell wall protein is selected from the group consisting of Aga1 claim 7 , Aga2 claim 7 , Agα1 claim 7 , Cwp1 claim 7 , Cwp2 claim 7 , Gas1p claim 7 , Yap3p claim 7 , Flo1p claim 7 , Crh2p claim 7 , Pir1 claim 7 , Pir2 claim 7 , Pir3 claim 7 , and Pir4.9. The host cell of claim 1 , wherein each of the PDZ Domain and the PDZ-binding peptide comprise a Cys residue.10. The host cell of claim 1 , wherein the polynucleotide of part (a) further encodes an enhancer domain.11. The host cell of claim 10 , wherein the enhancer domain is a variant of the 10fibronectin type III domain of human fibronectin (FN3).12. The host cell of claim 1 , wherein the PDZ Domain is selected from the group consisting of an InaD PDZ domain (SEQ ID NO: 2) claim 1 , a Dishevelled 1-like ( ...

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Номер записи: 82
06-02-2014 дата публикации

Displaying native human IgE neutralizing FceRla-contacting IgE B-cell epitopes by constraining super beta(b)-strands and cystine knots on thermostable protein scaffold

Номер: US20140039162A1
Автор: Chen Swy-Shen
Принадлежит:

Vaccine displaying native antigenic loops of immunoglobulin E is critical for eliciting neutralizing anti-IgE antibodies. The embodiment of the invention enables the display of native antigenic IgE receptor-contacting loops as IgE B-cell vaccines via three steps of constraining methods. The loops of multiple antigenic B-cell epitopes can be molecularly grafted in, and conformationally constrained by the energy favorable flanking beta (b)-stands, i.e., the super b-strands identified in this invention. The constrained loops can be further stabilized in replacing a selective loop within the cystine knot peptide. These dual constrained antigenic loops are then integrated onto thermostable protein scaffolds, folded in the oxidative milieu that provides further conformational constraint and high yield. 1. A method of identifying the scaffolding super beta-strands for the FG loop sequences of the human immunoglobulin E constrained on a protein scaffold , wherein amino acids from the flanking N-terminus and C-terminus are truncated , and wherein the FG loop presented by truncated beta-strands on N- and C-termini is immunoreactive with neutralizing anti-IgE in non-denaturing conditions , and wherein the FG loop within the flanking beta-strands constrained on the protein scaffold is an IgE B-cell vaccine candidate , whereby neutralizing anti-IgE is elicited in allergic patients , neutralize circulating and mucosal IgE in patients.2. The method of claim 1 , wherein the scaffolding super beta-strands are derived from the sequences the flanking super beta-strand sequences: YQCRVT and LMPST and the 3′ sequence of TKTSGPR critical for the core FG loop native sequence claim 1 , HLPR.3. The methods of claim 1 , wherein the loop sequence of the high affinity IgE receptor binding C2-3 loop claim 1 , BC claim 1 , and DE are exchanged with the native FG loop sequence claim 1 , and wherein the swapped loop of C2-3 claim 1 , BC claim 1 , and DE human IgE sequence claim 1 , respectively ...

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Номер записи: 83
13-02-2014 дата публикации

Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use

Номер: US20140046030A1
Автор: Alex STEINER, Avinash GILL, Chris Murray, Christopher D. Thanos, Gang Yin, Kalyani Penta, Leslie Mcevoy, Ramesh Baliga, Sonia Pollitt, Sunil Bajad
Принадлежит: Sutro Biopharma Inc

Provided herein are antibodies comprising non-natural amino acid residues at site-specific positions, compositions comprising the antibodies, methods of their production and methods of their use. The antibodies are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

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Номер записи: 84
20-02-2014 дата публикации

Process for Reduction and/or Removal of FXI and FXIa from Solutions Containing said Coagulation Factors

Номер: US20140051839A1
Автор: Bal Frederic, Gruber Gerhard, Marks Frank, Schultz Petra, Winge Stefan
Принадлежит: Octapharma AG

A process for reduction and/or removal of FXI and FXIa from a source solution containing said coagulation factors and as main components immunoglobulins comprising the following steps: 1) A process for reduction and/or removal of FXI and FXIa from a source solution containing said coagulation factors and as main components immunoglobulins comprising the following steps:a) contacting the FXI and/or FXIa containing solution with an affinity chromatographic gel wherein heparin or heparan is linked to the matrix material;b) allowing adsorption of FXI and/or FXIa andc) separation of the liquid deprived of FXI and/or FXIa from the adsorption media.2) The process according to wherein the active sites of the affinity chromatography gel are either already saturated or are allowed to saturate with antithrombin.3) The process according to wherein silicates selected from the group of silica claim 1 , perlites claim 1 , zeolithes or diatomaceous earth are additionally used as adsorbens of FXI and/or FXIa.4) The process according to wherein an additional adsorption medium selected from the group of aluminium hydroxide claim 1 , aluminium oxide hydroxide or aluminium oxide is used.5) The process according to wherein one adsorption on heparin or heparan linked to a matrix material is performed after the chromatographic material was preconditioned with Antithrombin-III.6) The process according to wherein the source solution primarily contains immunoglobulin-γ.7) The process according to wherein the liquid deprived of FXI and/or FXIa is further subjected to at least one virus inactivation step selected from solvent/detergent treatment claim 1 , UV-radiation claim 1 , pasteurization claim 1 , low pH incubation claim 1 , caprylate precipitation or nanofiltration to obtain a virus inactivated solution deprived of FXI and/or FXIa.8) The process according to wherein the claim 1 , optionally virus inactivated claim 1 , liquid deprived of FXI and/or FXIa is concentrated to obtain a ...

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Номер записи: 85
06-03-2014 дата публикации

Avian Transgenesis and Exogenous Proteins

Номер: US20140065690A1
Автор: Harvey Alex J., Ivarie Robert D., Liu Guodong, Morris Julie A., Rapp Jeffrey C.
Принадлежит:

This invention provides vectors and methods for the stable introduction of exogenous nucleic acid sequences into the genome of a bird and for expressing said exogenous sequences to alter the phenotype of the bird or to produce desired proteins. In particular, transgenic chickens are produced which express exogenous sequences in their oviducts. Eggs which contain exogenous proteins are also produced. 1. An exogenous protein produced in a tubular gland cell of a fully transgenic avian comprising an exogenous nucleotide sequence encoding said exogenous protein operably linked to a promoter in the genome.2. The exogenous protein of claim 1 , wherein said protein is contained in egg white of an egg of the transgenic avian.3. The exogenous protein of claim 1 , wherein said protein is obtained from a hard shell egg laid by the transgenic avian.4. The exogenous protein of claim 1 , wherein said transgenic avian is a chicken.5. The exogenous protein of claim 1 , wherein said promoter is a tissue specific promoter.6. The exogenous protein of claim 5 , wherein said tissue specific promoter is an oviduct specific promoter.7. The exogenous protein of claim 6 , wherein said oviduct specific promoter is an ovalbumin promoter.8. The exogenous protein of claim 1 , wherein said protein is a human protein.9. The exogenous protein of claim 1 , wherein said protein is an antibody.10. The exogenous protein of claim 1 , wherein said protein is an enzyme. This application is a continuation of application Ser. No. 11/274,674, filed Nov. 15, 2005, which is a continuation of application Ser. No. 10/696,671, filed Oct. 28, 2003, now U.S. Pat. No. 7,521,591, which is a continuation of application Ser. No. 09/173,864, filed Oct. 16, 1998, now U.S. Pat. No. 6,730,822, issued May 4, 2004, which claims the benefit of U.S. Provisional Application No. 60/062,172, filed Oct. 16, 1997. The entire teachings of each of the above applications are expressly incorporated herein by reference.a) Field of the ...

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Номер записи: 86
06-03-2014 дата публикации

Methods to control protein heterogeneity

Номер: US20140065710A1
Автор: Cornelia BENGEA, Lisa M. Rives, Xiaobei Zeng
Принадлежит: AbbVie Inc

The instant invention relates to the field of protein production, and in particular to compositions and processes for controlling and limiting the heterogeneity of proteins expressed in host cells.

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Номер записи: 87
06-03-2014 дата публикации

Multivalent Heteromultimer Scaffold Design and Constructs

Номер: US20140066378A1
Автор: "DANGELO Igor Edmondo Paolo", DIXIT Surjit Bhimarao, NG Gordon Yiu Kon, SANCHES Mario
Принадлежит:

Provided herein are multifunctional heteromultimer proteins. In specific embodiments is a heteromultimer comprising: at least two polypeptide constructs, each polypeptide construct comprising at least one cargo polypeptide attached to a transporter polypeptide, said transporter polypeptides derived from a monomeric native protein such that said monomeric constructs associate to form the heteromultimer and said transporter polypeptides associate to form a quasi-native structure of the monomeric native protein or analog thereof. These therapeutically novel molecules encompass heteromultimers comprising constructs that function as scaffolds for the conjugation or fusion of therapeutic molecular entities (cargo polypeptides) resulting in the creation of bispecific or multivalent molecular species. Provided herein is a method for creation of bispecific or multivalent molecular species. 1. A heteromultimer comprising:a first polypeptide construct that comprises (i) a first transporter polypeptide; anda second polypeptide construct that comprises (ii) a second transporter polypeptide;wherein each of said first and second transporter polypeptide comprises an amino acid sequence with at least 90% identity to a segment of an albumin polypeptide; andwherein said first and second transporter polypeptides are obtained by segmentation of said albumin polypeptide at a segmentation site, such that the segmentation results in a deletion of zero to 3 amino acid residues at the segmentation site,wherein said transporter polypeptides self-assemble to form a quasi-native structure of the monomeric form of said albumin polypeptide.2. The heteromultimer of claim 1 , wherein a) the segmentation site resides on a loop of the albumin polypeptide that has a high solvent accessible surface area (SASA) and limited contact with the rest of the albumin structure claim 1 , b) the segmentation results in a complementary interface between the transporter polypeptides claim 1 , wherein the interface ...

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Номер записи: 88
06-03-2014 дата публикации

Modified Amino Acids

Номер: US20140066598A1
Автор: Stafford Ryan, Thanos Christopher D., Yang Wenjin
Принадлежит: Sutro Biopharma, Inc.

Provided herein are modified amino acids comprising an azido group, polypeptides, antibodies and conjugates comprising the modified amino acids, and methods of producing the polypeptides, antibodies and conjugates comprising the modified amino acids. The polypeptides, antibodies and conjugates are useful in methods of treatment and prevention, methods of detection and methods of diagnosis. 2. The compound of claim 1 , wherein D is —Ar—W—.3. The compound of claim 1 , wherein D is —W—Y—C(O)—Y—W—.7. The compound of wherein:{'sub': 1', '1', '2', '2, 'D is —W—Y—C(O)—Y—W—; and'}{'sub': '1', 'each Yis independently —NH— or —O—.'}8. The compound of claim 1 , wherein:{'sub': 1', '1', '2', '2, 'D is —W—Y—C(O)—Y—W—;'}{'sub': '2', 'each Yis independently an N-linked or C-linked pyrrolidinylene; and'}{'sub': '2', 'each Wis a single bond.'}9. The compound of claim 1 , wherein:{'sub': 1', '1', '2', '2, 'D is —W—Y—C(O)—Y—W—;'}{'sub': '2', 'each Yis independently a single bond, —NH— or —O—; and'}{'sub': '2', 'each Wis lower alkylene.'}11. The compound of claim 1 , wherein each of Wand Wis independently C-Calkylene.15. A polypeptide comprising an amino acid residue corresponding to the compound of .16. A conjugate comprising the polypeptide of linked to a payload and optionally comprising a linking moiety between the polypeptide and the payload.17. An antibody comprising an amino acid residue corresponding to the compound of .18. A conjugate comprising the antibody of linked to a payload and optionally comprising a linking moiety between the antibody and the payload.19. An orthogonal tRNA aminoacylated with an amino acid residue corresponding to the compound of .20. A method of producing a polypeptide claim 19 , comprising contacting a polypeptide with the orthogonal tRNA of under conditions suitable for incorporating the amino acid residue into the polypeptide.21. The method of claim 20 , wherein the orthogonal tRNA base pairs with a codon that is not normally associated with an ...

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Номер записи: 89
06-03-2014 дата публикации

ALPHABODY LIBRARIES AND METHODS FOR PRODUCING THE SAME

Номер: US20140066601A1
Автор: Desmet Johan, Henderikx Maria, Lasters Ignace, Wehnert Anita
Принадлежит:

The invention provides single-chain Alphabody library comprising at least 100 different-sequence single-chain Alphabody polypeptides, wherein said Alphabody polypeptides differ from each other in at least one of a defined set of 5 to 20 variegated amino acid residue positions, and wherein at least 70% but not all of said variegated amino acid residue positions are located either in the loop, helix surface or linker region of the Alphabody. The invention further provides methods for use of the Alphabody libraries and Alphabodies obtainable by the methods of the invention. 1. A single-chain Alphabody library comprising at least 100 different-sequence single-chain Alphabody polypeptides , wherein said Alphabody polypeptides differ from each other in at least one of a defined set of 5 to 20 variegated amino acid residue positions , and wherein at least 70% but not all of said variegated amino acid residue positions are located either:(i) at heptad e-positions in a first alpha-helix of the Alphabody polypeptides and at heptad g-positions in a second alpha-helix, and optionally at heptad b-positions in said first alpha-helix of the Alphabody polypeptides and/or at heptad c-positions in said second alpha-helix of the Alphabody polypeptides, or(ii) at heptad b-, c- and f-positions in one alpha-helix of the Alphabody polypeptides, or(iii) at positions in a linker fragment connecting two consecutive alpha-helices of the Alphabody polypeptides.2. The single-chain Alphabody library according to claim 1 , wherein the constant claim 1 , non-variegated part of each of said at least 100 different-sequence single-chain Alphabody polypeptides is of non-natural origin.3. The single-chain Alphabody library according to claim 1 , wherein said variegated amino acid residue positions are exclusively occupied by naturally occurring amino acid types.4. A mixture of Alphabody libraries claim 1 , comprising between two and six of said different constituting libraries claim 1 , wherein each ...

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Номер записи: 90
06-03-2014 дата публикации

Methods for Genetic Diversification in Gene Conversion Active Cells

Номер: US20140068795A1
Автор: Hiroshi Arakawa, Jean-Marie Buerstedde
Принадлежит: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH

The invention relates to a modified lymphoid cell having gene conversion fully or partially replaced by hypermutation, wherein said cell has no deleterious mutations in genes encoding paralogues and analogues of the RAD51 protein, and wherein said cell is capable of directed and selective genetic diversification of a target nucleic acid by hypermutation or a combination of hypermutation and gene conversion. The invention also relates to a method for diversifying any transgenic target gene in said cell. Preferably, the target gene is integrated into the immunoglobulin light or heavy chain locus by targeted integration.

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Номер записи: 91
13-03-2014 дата публикации

Immunoglobulin Constructs Comprising Selective Pairing of the Light and Heavy Chains

Номер: US20140072581A1
Автор: "DANGELO Igor Edmondo Paolo", DIXIT Surjit Bhimarao, NG Gordon Yiu Kon, UROSEV Dunja
Принадлежит: Zymeworks Inc.

Disclosed herein is an isolated immunoglobulin construct comprising a first monomeric polypeptide comprising a first single chain Fv polypeptide connected to a first constant domain polypeptide; and a second monomeric polypeptide comprising a second single chain Fv polypeptide, connected to a second constant domain polypeptide; each said constant domain polypeptide comprising at least one each of a CL domain, a CH1 domain, a CH2 domain and a CH3 domain or fragments, variants or derivatives thereof; and wherein said first and second constant domain polypeptide form a Fc region. 1. An immunoglobulin construct comprising:a single chain Fab region (scFab) comprising:a variable region polypeptide (VH) from an immunoglobulin heavy chain,a variable region polypeptide (VL) from an immunoglobulin light chain,a constant region polypeptide (CL) from an immunoglobulin light chain, anda constant region polypeptide (CH1) from an immunoglobulin heavy chain;wherein said VH and VL are connected by a first linker to form a single chain Fv construct (scFv).2. The immunoglobulin construct of claim 1 , wherein said CL and CH1 are connected by a second linker.3. The immunoglobulin construct of claim 2 , wherein said single chain Fab region has a sequence comprising VH-L1-VL-CL-L2-CH1 claim 2 , wherein L1 and L2 are first and second linkers.4. The immunoglobulin construct of claim 2 , wherein the single chain Fab region has a sequence comprising VH-L1-VL-L3-CL-L2-CH1 claim 2 , wherein L1 claim 2 , L2 and L3 are linkers.5. The immunoglobulin construct of claim 2 , wherein the single chain Fab region has a sequence comprising VL-L4-VH-CH1-L5-CL claim 2 , wherein L4 and L5 are linkers.6. The immunoglobulin construct of wherein each linker is a polypeptide comprising from about 1 to about 100 amino acids.7. The immunoglobulin construct of claim 6 , wherein said linker comprises an amino acid sequence comprising amino acids selected from Gly (G) claim 6 , Ser (S) and Glu (E).8. The ...

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Номер записи: 92
13-03-2014 дата публикации

ASYMMETRIC POROUS ADSORPTIVE BEAD

Номер: US20140073769A1
Автор: Andrew Lyddiatt, Aquino Dennis, Bian Nanying, Cheng Kwok-Shun, Gagnon Brian, Ramaswamy Senthilkumar, Soice Neil, Umana Joaquin A., Wang Chen
Принадлежит: EMD MILLIPORE CORPORATION

The present invention relates to an asymmetric chromatography media suitable for separations applications, particularly as packed bed, fluidized bed or magnetized bed chromatography media. In certain embodiments, the asymmetric chromatography media comprises asymmetric particles, preferably beads, having at least two distinct, controlled pore size distributions. Preferably one of the distinct pore size distributions is in an internal region of the particle, and the other is in an external region or coating on the particle. These distinct pore size distributions can be modified with uniform or alternatively unique functional groups or mixtures of functional groups. The present invention allows for the control over pore size distribution within an asymmetric porous particle by providing a distinct internal region, preferably in the form of a bead, and a distinct external region, preferably in the form of a coating on the bead. 1. A method of separating a monoclonal antibody from a host cell protein , the method comprising contacting a mixture of antibody and host cell protein with an asymmetric chromatography particle comprising an internal region and an external region , the internal region and the external region having different one or more characteristics selected from the group consisting of pore size , wherein the monoclonal antibody is absorbed onto the external region and the host cell protein is adsorbed onto the internal region.2. The method of claim 1 , wherein the characteristic is pore size.3. The method of claim 1 , wherein the internal region has a smaller pore size than the external region.4. The method of claim 1 , wherein the external region comprises a thickness of 1 to 15 microns.5. The method of claim 1 , wherein the chromatography particle is modified with a hydrophilic ligand carrier.6. The method of wherein the chromatography particle is modified with a hydrophilic ligand carrier wherein the hydrophilic ligand carrier is dextran. The present ...

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Номер записи: 93
20-03-2014 дата публикации

Harvest operations for recombinant proteins

Номер: US20140080180A1
Автор: Bradley R. Snedecor, Deepa Nadarajah, Jane V. Gunson, Kim Kaleas, Michael W. Laird, Rachel Adams, Richard St. John
Принадлежит: Genentech Inc

The present invention contemplates methods of producing a recombinant protein comprising fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, harvesting said recombinant protein under conditions where dO 2 levels are greater than 0%, purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm. Furthermore, method of producing a recombinant protein comprising fermenting a menE gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, harvesting said recombinant protein, purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm, wherein the recombinant protein yield is increased by about 20% or greater is contemplated.

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Номер записи: 94
27-03-2014 дата публикации

Biomarkers for Assessment of the Molecular Quality in Biospecimens

Номер: US20140087401A1
Автор: Alexander Oliver VORTMEYER, Jie Li
Принадлежит: Individual

The present invention provides compositions and methods for assessing the amount of degradation in a biospecimen. The invention relates to the discovery that dynamic changes in protein biomarkers correlate to the amount of degradation in a biospecimen. The present invention also provides kits for assessing the amount of degradation in a biospecimen.

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Номер записи: 95
03-04-2014 дата публикации

Methods of generating libraries and uses thereof

Номер: US20140094392A1
Автор: Andrew B. Cubitt, Peter M. Bowers, Robert A. Horlick
Принадлежит: Anaptysbio Inc

This invention relates to methods for the generation of humanized antibodies, particularly a humanized antibody heavy chain protein and a humanized antibody light chain protein. The method comprises using cells that express or can be induced to express Activation Induced Cytidine Deaminase (AID).

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Номер записи: 96
10-04-2014 дата публикации

COMPOSITIONS AND METHODS FOR ENHANCING PRODUCTION OF A BIOLOGICAL PRODUCT

Номер: US20140099666A1
Автор: Bettencourt Brian, Hinkle Gregory, Hogan Shannon, Kocisko David, Manoharan Muthiah, Maraganore John M., Pollard Stuart, Rossomando Anthony
Принадлежит: ALNYLAM PHARMACEUTICALS, INC.

The invention provides compositions and methods for producing a biological product from a host cell. In various embodiments, the biological product is a polypeptide, a metabolite, a nutraceutical, a chemical intermediate, a biofuel, a food additive, or an antibiotic. In one aspect, the invention provides for a method for producing a biological product from a host cell. The method generally comprises contacting the cell with a RNA effector molecule, a portion of which is complementary to a target gene, maintaining the cell in a large-scale bioreactor for a time sufficient to modulate expression of the target gene, wherein the modulation enhances production of the biological product from the cell, and isolating the biological product from the cell. 1. A method for producing a biological product in a large scale host cell culture , comprising:(a) contacting a host cell in a large scale host cell culture with at least a first RNA effector molecule, a portion of which is complementary to at least one target gene of a host cell,(b) maintaining the host cell culture for a time sufficient to modulate expression of the at least one first target gene, wherein the modulation of expression improves production of a biological product in the host cell;(c) isolating the biological product from the host cell;wherein the large scale host cell culture is at least 1 liter in size, and wherein the host cell is contacted with at least a first RNA effector molecule by addition of the RNA effector molecule to a culture medium of the large scale host cell culture such that the target gene expression is transiently inhibited.2. (canceled)3. The method of claim 1 , wherein the host cell in the large scale host cell culture is contacted with a plurality of RNA effector molecules claim 1 , wherein the plurality of RNA effector molecules modulate expression of at least one target gene claim 1 , at least two target genes claim 1 , or a plurality of target genes.5. The method of claim 4 , wherein ...

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Номер записи: 97
07-01-2021 дата публикации

Transgenic chicken that produces human antibodies

Номер: US20210000088A1
Автор: Philip A. Leighton, William Don Harriman
Принадлежит: Crystal Bioscience Inc

A transgenic chicken having a genome comprising a modified immunoglobulin heavy chain (IgH) locus is provided. The locus lacks the entire contiguous endogenous chicken V-D-J region and contains a human VH segment, a human D cluster, a human J segment and a plurality of upstream pseudogenes based on human VH sequences. The modified IgH locus undergoes V(D)J recombination in the chicken and the chicken produces antibodies that have a diversified immunoglobulin heavy chain.

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Номер записи: 98
07-01-2021 дата публикации

ENHANCED IMMUNOGLOBULIN DIVERSITY

Номер: US20210000089A1
Автор: DUONG Bao, Mueller Werner, WABL Matthias
Принадлежит:

The invention provides compositions and methods for enhanced production of immunoglobulin diversity. Specifically, the invention provides compositions and methods for making accessible a B cell receptor repertoire that has not been culled by developmental tolerance mechanisms. The invention also provides transgenic animals, cells, and antibodies resulting from these compositions and methods. 1. A genetically modified mouse comprising:{'sub': H', 'H', 'H, '(a) a first immunoglobulin heavy chain allele, comprising V, D and Jgene segments and one or more exons encoding immunoglobulin constant domains, in which a first expression cassette comprising a first splice acceptor and a first stop codon is inserted in antisense orientation with respect to transcriptional direction downstream of the first immunoglobulin heavy chain allele Jgene segments and upstream of the first immunoglobulin heavy chain allele exons encoding constant domains, wherein the first cassette is flanked by site-specific recognition sequences; and'}{'sub': H', 'H', 'H, '(b) a second immunoglobulin heavy chain allele comprising V, D and Jgene segments and one or more exons encoding immunoglobulin constant domains, in which a second expression cassette comprising a second splice acceptor and a second stop codon is inserted in sense orientation with respect to transcriptional direction downstream of the second immunoglobulin heavy chain Jgene segments and upstream of the second immunoglobulin heavy chain allele exons encoding constant domains, wherein the second cassette is flanked by site-specific recognition sequences,'}wherein the first immunoglobulin heavy chain allele is capable of expressing a functional first immunoglobulin heavy chain and the second immunoglobulin heavy chain allele can undergo productive VDJ rearrangement but is deficient in expression of a functional second immunoglobulin heavy chain, wherein expression of the first allele can be inactivated and the deficiency in expression of ...

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Номер записи: 99
06-01-2022 дата публикации

PRODUCTION OF HETEROMULTIMERIC PROTEINS USING MAMMALIAN CELLS

Номер: US20220002386A1
Автор: NG Domingos, SCHEER JUSTIN, SHATZ WHITNEY
Принадлежит: Genentech, Inc.

Described herein are methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins) capable of specifically binding to more than one target. The targets may be, for example, different epitopes located on a single molecule or located on different molecules. 164-. (canceled)65. A method of preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain , wherein the first hinge-containing polypeptide is associated with a first light chain , and ii) a second hinge-containing polypeptide having a second heterodimerization domain , wherein the second hinge-containing polypeptide is associated with a second light chain , wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface , and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond , the method comprising the steps of:(a) culturing a first host cell capable of expressing a first hinge-containing polypeptide and a first light chain;(b) culturing a second host cell capable of expressing a second hinge-containing polypeptide and a second light chain; and,(c) obtaining a combined culture medium for the first host cell and the second host cell without disrupting cell membrane of the first and second host cells, wherein the combined culture medium comprises the heteromultimeric protein, and wherein the first host cell and the second host cell are each a mammalian cell.66. The method of claim 65 , wherein obtaining the combined culture medium comprises: (2) harvesting a second culture medium for the second host cell; and', '(3) combining the first culture medium and the second culture medium to obtain the combined culture medium, or, '(A) (1) harvesting a first culture medium for the first host cell;'}(B) harvesting culture medium of a combined cell culture ...

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Номер записи: 100