БИОТЕХНОЛОГИЧЕСКОЕ ПОЛУЧЕНИЕ LNT, LNNT И ИХ ФУКОЗИЛИРОВАННЫХ ПРОИЗВОДНЫХ

Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans

Use of recombinant enzymes for the preparation of GDP-L-fucose and fucosylated glycans is disclosed. GDP-L-fucose functions as a fucose donor in the biosynthetic route leading to the fucosylated glycans, which have therapeutic utility. A process for preparing GDP-L-fucose and fucosylated glycans, and means useful in the process are provided. Said means include enzymes, chimeric enzymes, DNA sequences, genes, vectors and host cells. An assay for the determination of GDP-fucose and fucosyl-transferase, and a test kit therefore are also provided.

BIOTECHNOLOGICAL PRODUCTION OF LNT, LNNT AND THE FUCOSYLATED DERIVATIVES THEREOF

The invention relates to primarily genetically engineered microorganisms for the in-vivo synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and the fucosylated derivatives thereof, and to uses of said microorganisms in methods for producing lacto-N-tetraose and lacto-N-neotetraose and the fucosylated derivatives thereof.

Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans

Use of recombinant enzymes for the preparation of GDP-L-fucose and fucosylated glycans is disclosed. GDP-L-fucose functions as a fucose donor in the biosynthetic route leading to the fucosylated glycans, which have therapeutic utility. A process for preparing GDP-L-fucose and fucosylated glycans, and means useful in the process are provided. Said means include enzymes, chimeric enzymes, DNA sequences, genes, vectors and host cells. An assay for the determination of GDP-fucose and fucosyltransferase, and a test kit therefore are also provided.

NUCLEIC ACIDS ENCODING GDP-FUCOSE PYROPHOSPHORYLASE

DNA sequences encoding GDP-fucose pyrophosphorylase (GTP + fucose-1-PGDP-fucose + PPi), are provided. The enzymes can be used in the synthesis of desired carbohydrate structures.

ＬＮＴ, ＬＮＮＴ 및 이들의 푸코실화 유도체의 생명공학적 생산

... 본 발명은 주로 락토-N-테트라오스 (LNT) 및 락토-N-네오테트라오스 (LNnT), 및 이들의 푸코실화 유도체의 인-비보 합성을 위한 유전자 조작 미생물, 및 락토-N-테트라오스 및 락토-N-네오테트라오스 및 이들의 푸코실화 유도체의 생산 방법에서의 상기한 미생물의 용도에 관한 것이다.

RECOMBINANT E. COLI FOR PRODUCING FUCOSYLLACTOSE, AND METHOD FOR PRODUCING FUCOSYLLACTOSE BY USING SAME

The present invention relates to recombinant E. coli for producing fucosyllactose and to a method for producing fucosyllactose by using the same. The recombinant E. coli has one or more pulverized genes for coding fucose isomerase (FucI), fuculose kinase (FucK), and fuculose 1-phosphate aldolase (FucA), which are fucose metabolism enzymes; and contains a lac operon consisting of a lacZ gene, a wild-type lacY gene, and a wild-type lacA gene for coding beta-galactosidase having a lower activity than wild-type beta-galactosidase, or a lac operon consisting of only a wild-type lacY gene and a wild-type lacA gene without a wild-type lacZ gene, instead of a wild-type lac operon. According to the present invention, 2- or 3-fucosyllactose can be produced at high productivity. COPYRIGHT KIPO 2016 ...

Biotechnological production of LNT, LNnT and the fucosylated derivatives thereof

The invention relates to primarily genetically engineered microorganisms for the in-vivo synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and the fucosylated derivatives thereof, and to uses of said microorganisms in methods for producing lacto-N-tetraose and lacto-N-neotetraose and the fucosylated derivatives thereof.

micro-organismo geneticamente modificado, uso de um micro-organismo geneticamente modificado, e, método para preparar lacto-n-tetraose ou lacto-n-neotetraose ou um derivado fusosilado de lacto-n-tetraose ou lacto-n-neotetraose

NUCLEIC ACIDS ENCODING GDP-FUCOSE PYROPHOSPHORYLASE

DNA sequences encoding GDP-fucose pyrophosphorylase (GTP + fucose-1-P→GDP-fucose + PPi), are provided. The enzymes can be used in the synthesis of desired carbohydrate structures.

Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans

Use of recombinant enzymes for the preparation of GDP-L-fucose and fucosylated glycans is disclosed. GDP-L-fucose functions as a fucose donor in the biosynthetic route leading to the fucosylated glycans, which have therapeutic utility. A process for preparing GDP-L-fucose and fucosylated glycans, and means useful in the process are provided. Said means include enzymes, chimeric enzymes, DNA sequences, genes, vectors and host cells. An assay for the determination of GDP-fucose and fucosyl-transferase, and a test kit therefore are also provided.

A YEAST RECOMBINANT CELL CAPABLE OF PRODUCING GDP-FUCOSE

A yeast strain expressing a bi-functional fucokinase/GDP-L-fucose pyrophosphorylase enzyme and capable of producing GDP-L-fucose in vivo is provided. Also provided are yeast cells which express a GDP-L-fucose transporter and /or a fucosyl transferase with the bi-functional enzyme. In addition, the said yeast contains one or more expression cassettes for fusion proteins of heterologous glycosylation pathway and an ER/Golgi retention sequence. Finally, the invention also provides a method for producing recombinant target glycoproteins.

BIOTECHNOLOGICAL PRODUCTION OF LNT, LNNT AND THE FUCOSYLATED DERIVATIVES THEREOF

NUCLEIC ACIDS ENCODING GDP-FUCOSE PYROPHOSPHORYLASE

DNA sequences encoding GDP-fucose pyrophosphorylase (GTP + fucose-1-P}GDP-fucose + PPi), are provided. The enzymes can be used in the synthesis of desired carbohydrate structures.

NUCLEIC ACIDS ENCODING GDP-FUCOSE PYROPHOSPHORYLASE

FERMENTATIVE PRODUCTION

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of metabolically engineered cells and use of said cell in a cultivation, preferably a fermentation. The present invention describes a cell for the production of a compound. The cell comprises a pathway for the production of the compound, which can be a disaccharide, oligosaccharide and/or a Neu(n)Ac-containing bioproduct, wherein (n) is 4, 5, 7, 8 or 9 or a combination thereof. The cell is metabolically engineered for enhanced synthesis of acetyl-Coenzyme A. The invention also resides in a method of producing such compound by cultivation, preferably a fermentation, with such a cell.

ENZYMATIC METHOD FOR PREPARATION OF GDP-FUCOSE

Method for preparing 2'-Fucosyllactose from genetically-engineered bacteria

Nucleic acid encoding GDP-fucose pyrophosphorylase

GDP―フコースピロホスホリラーゼをコードする核酸

GDP-フコースピロホスホリラーゼ(GTP+フコース-1-P→GDP-フコース+PPi)をコードするDNA配列が供給される。その酵素は、所望する炭水化物構造体の合成に使用され得る。 ...

Biotechnological production of LNT, LNnT and the fucosylated derivatives thereof

BIOTECHNOLOGICAL PRODUCTION OF LNT, LNnT AND THE FUCOSYLATED DERIVATIVES THEREOF

Biotechnological production of LNT, LNnT and the fucosylated derivatives thereof The invention relates to primarily genetically engineered microorganisms for the in-vivo synthesis of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and the fucosylated derivatives thereof, and to uses of said microorganisms in methods for producing lacto-N-tetraose and lacto-N-neotetraose and the fucosylated derivatives thereof. [No suitable figure] ...

BIOTECHNOLOGICAL PRODUCTION OF LNT, LNNT AND THE FUCOSYLATED DERIVATIVES THEREOF

The present invention relates to primarily genetically modified microorganisms for in vivo synthesis of lacto-N-tetrose (LNT) and lacto-N-neotetrose (LNnT), and their fucosylated derivatives, and to uses of such microorganisms in methods of producing lacto-N-tetrose and lacto-N-neotetrose, and their fucosylated derivatives.

Use of recombinant enzymes for preparing GDP-L-fucose and fucosylated glycans

Use of recombinant enzymes for the preparation of GDP-L-fucose and fucosylated glycans is disclosed. GDP-L-fucose functions as a fucose donor in the biosynthetic route leading to the fucosylated glycans, which have therapeutic utility. A process for preparing GDP-L-fucose and fucosylated glycans, and means useful in the process are provided. Said means include enzymes, chimeric enzymes, DNA sequences, genes, vectors and host cells. An assay for the determination of GDP-fucose and fucosyltransferase, and a test kit therefore are also provided.

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of treating a patient who has cancer , comprising administering to the patient an effective amount of an antibody specifically binding to an MHC class I or II molecule complexed with an antigen consisting of the amino acid sequence of VQLDSIEDLEV (SEQ ID NO: 32) ,wherein said cancer is selected from the group consisting of glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer, head- and neck squamous cell carcinoma, and uterine cancer.2. The method of claim 1 , wherein the antibody is a polyclonal antibody claim 1 , a monoclonal antibody claim 1 , a bi-specific antibody claim 1 , or a chimeric antibody.3. The method of claim 1 , wherein the antibody binds to the HLA-restricted antigen with a binding affinity of below 20 nanomolar.4. The method of claim 1 , wherein the antibody binds to the MHC class I molecule complexed with the HLA-restricted antigen.5. The method of claim 1 , wherein the antibody is humanized.6. The ...

Variants of a DNA Polymerase of the Polx Family

The invention relates to variants of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand, or of a functional fragment of such a polymerase, comprising at least one mutation of a residue in at least one specific position, and to uses of said variants, in particular for the synthesis of nucleic acid molecules comprising 3′-OH modified nucleotides. 124-. (canceled)25. A DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand , said DNA polymerase comprising at least one mutation of a residue in at least one position selected from the group consisting of E457 , T331 , G332 , G333 , F334 , R336 , K338 , H342 , D343 , V344 , D345 , F346 , A397 , D399 , D434 , V436 , A446 , L447 , L448 , G449 , W450 , G452 , R454 , Q455 , F456 , R458 , R461 , N474 , E491 , D501 , Y502 , 1503 , P505 , R508 , N509 and A510 , the positions indicated being determined by alignment with SEQ ID No. 1.26. The DNA polymerase of the polX family according to claim 25 , said DNA polymerase being capable of synthesizing a DNA strand and/or an RNA strand.27. The DNA polymerase of the polX family according to claim 25 , said DNA polymerase being a variant of Pol IV claim 25 , Pol μ claim 25 , or of the terminal deoxyribonucleotidyl transferase (TdT).28. The DNA polymerase of the polX family according to claim 25 , in which at least one mutation consists of a substitution claim 25 , a deletion or an addition of one or more amino acid residues.29. The DNA polymerase of the polX family according to claim 25 , said DNA polymerase comprising at least one mutation of a residue in at least one position selected from the group consisting of T331 claim 25 , G332 claim 25 , G333 claim 25 , F334 claim 25 , R336 claim 25 , D343 claim 25 , L447 claim 25 , L448 claim 25 , G449 claim 25 , W450 claim 25 , G452 claim 25 , R454 claim 25 , Q455 claim 25 , E457 claim 25 , R461 and R508.30. The DNA polymerase of the ...

MODIFIED TEMPLATE-INDEPENDENT ENZYMES FOR POLYDEOXYNUCLEOTIDE SYNTHESIS

The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences. 1. A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation selected from the group consisting of E33K , E180L , E180K , M192E , M192K , M192W , W303H , L381K , L381Q , L381R , L381V , W450H , R454I , R454T , R454K , E457K , R461V , R461Q , R461V , N474R , and N474K , said modified TdT capable of adding a nucleotide analog comprising a removable blocking moiety at a 3′-Oxygen of the analog to a 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.2. The modified TdT of claim 1 , comprising a mutation E457K.3. The modified TdT of claim 1 , comprising the mutations E180K claim 1 , M192W claim 1 , L381R claim 1 , and W450H.4. The modified TdT of claim 1 , comprising the mutations L381Q and W450H.5. The modified TdT of claim 1 , comprising the mutations E180L claim 1 , M193E claim 1 , L381K claim 1 , R461Q claim 1 , and N457K.6. The modified TdT of claim 1 , comprising the mutations E180K claim 1 , L381Q claim 1 , W450H and R461V.7. The modified TdT of claim 1 , comprising the mutations L381Q and W450H.8. The modified TdT of claim 1 , comprising the mutations E180L claim 1 , M192E claim 1 , L381K claim 1 , R461Q claim 1 , and N457K.9. The modified TdT of claim 1 , comprising the mutations E180K claim 1 , M192E claim 1 , L381K claim 1 , R454T claim 1 , and N47K.10. The modified TdT of claim 1 , comprising the mutations E180K claim 1 , M192K claim 1 , L381K claim 1 , R454T claim 1 , and N457R.11. The modified TdT of claim 1 , comprising the mutations E180K claim 1 , M192K claim 1 , L381K claim 1 , R454K claim 1 , ...

Variants of Terminal Deoxynucleotidyl Transferase and Uses Thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

Novel Peptides and Combination of Peptides for Use In Immunotherapy Against Various Cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of eliciting an immune response in a patient who has cancer , comprising administering to said patient a population of activated T cells that selectively recognize cells , which present a peptide consisting of the amino acid sequence of RLLGTEFQV (SEQ ID NO: 154) , wherein said cancer is selected from the group consisting of glioblastoma , breast cancer , colorectal cancer , renal cell carcinoma , chronic lymphocytic leukemia , hepatocellular carcinoma , non-small cell and small cell lung cancer , non-Hodgkin lymphoma , acute myeloid leukemia , ovarian cancer , pancreatic cancer , prostate cancer , esophageal cancer including cancer of the gastric-esophageal junction , gallbladder cancer and cholangiocarcinoma , melanoma , gastric cancer , urinary bladder cancer , head-and neck squamous cell carcinoma , and uterine cancer.2. The method of claim 1 , wherein the T cells are autologous to the patient.3. The method of claim 1 , wherein the T cells are obtained from a healthy donor.4. The method of claim 1 , wherein the T cells are obtained from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.5. The method of claim 1 , wherein the activated T cells are expanded in vitro.6. The method of claim 1 , wherein the peptide is in a complex ...

A Process for the Preparation of Nucleic Acid by Means of 3'-O-Azidomethyl Nucleotide Triphosphate

The invention relates to a method of nucleic acid synthesis comprising the use of 3′-O-azidomethyl blocked nucleotide triphosphates which comprises the step of adding a capping group to any uncleaved 3′-O-azidomethyl groups and to the use of kits comprising said capping groups in a method of nucleic acid synthesis. The invention also relates to capped nucleotide triphosphates and 3′-O-azidomethyl capping groups.

NOVEL USE

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis. 1. A method of nucleic acid synthesis comprising a step of providing a terminal deoxynucleotidyl transferase (TdT) enzyme comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 to 5 and 8 , such as any of SEQ ID NOs: 1 , 2 , or 8.2. (canceled)3. A method of nucleic acid synthesis , comprising steps of:(a) providing an initiator sequence;{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, '(b) adding a 3′-blocked nucleotide triphosphate to said initiator sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) as defined in ;'}(c) removal of all reagents from the initiator sequence;(d) cleaving the blocking group from the 3′-blocked nucleotide triphosphate in the presence of a cleaving agent; and(e) removal of the cleaving agent.4. The method as defined in claim 3 , wherein greater than 1 nucleotide is added by repeating steps (b) to (e).5. The method as defined in claim 3 , wherein the 3′-blocked nucleotide triphosphate is blocked by either a 3′-O-azidomethyl claim 3 , 3′-aminoxy or 3′-O-allyl group.6Saccharomyces cerevisiae.. The method as defined in claim 3 , wherein the terminal deoxynucleotidyl transferase (TdT) is added in the presence of an extension solution comprising one or more buffers claim 3 , such as Tris or cacodylate claim 3 , one or more salts; and/or inorganic pyrophosphatase claim 3 , such as purified claim 3 , recombinant inorganic pyrophosphatase from7. The method as defined in claim 3 , wherein step (b) is performed at a pH range of between 5 and 10 ...

Method for rescuing influenza virus and composition therefor

The present invention relates to a new method for rescuing an influenza virus and a composition therefor. The method comprises providing a host cell stably integrated with and expressing influenza virus PA, PB1, PB2 and NP genes, and introducing an influenza virus rescue system in which a stop codon is introduced into the PA, PB1, PB2 and NP genes respectively into the host cell to achieve virus rescue. The produced virus particles can be used as a live attenuated influenza vaccine, which is characterized in that, since the genes encoding the related proteins are mutated, it has no replication and proliferation ability in human and normal animal cells, and replication and proliferation can be achieved only in the host cells constructed above and it can fully stimulate the body immunity and effectively protect the body while ensuring the safety. 1. A method for rescuing an influenza virus , characterized in that a mammalian host cell stably expressing influenza virus PA , PB1 , PB2 and NP genes is provided , and an influenza virus rescue system comprising mutant PA , PB1 , PB2 and NP genes is introduced into the aforementioned host cell to achieve rescue , wherein the mutations make the influenza virus rescue system unable to rescue intact virus in natural mammalian cells.2. The method according to claim 1 , wherein the method comprises the following steps:(1) constructing a single- or multiple-plasmid system encoding the PA, PB1, PB2 and NP genes;(2) introducing the single- or multiple-plasmid system of step (1) into a mammalian cell, and screening a host cell stably expressing the four genes;(3) constructing recombinant plasmids for the mutant PA, PB1, PB2 and NP genes and recombinant plasmids encoding the four genes HA, NA, M and NS respectively to form an influenza virus rescue system, the mutations are achieved by introducing a TAG codon into each of the four gene sequences;(4) co-transfecting the influenza virus rescue system constructed in step (3) into the ...

SPATIAL CONTROL OF POLYNUCLEOTIDE SYNTHESIS BY STRAND CAPPING

Enzymatic polynucleotide synthesis with a template-independent polymerase is used to create multiple polynucleotides having different, arbitrary sequences on the surface of an array. The array provides a spatially-addressable substrate for solid-phase synthesis. Blocking groups are attached to the 3′ ends of polynucleotides on the array. Prior to polynucleotide extension, the blocking groups are removed at a selected location on the array. In an implementation, the blocking groups are acyl groups removed with a negative voltage created at an electrode. The array is then incubated with the polymerase and a single species of nucleotide. Nucleotides are incorporated onto the 3′ ends of the polynucleotides without blocking groups. Washing removes the polymerase and free nucleotides. To create polynucleotides with different sequences at different locations on the array, the location where the blocking groups are removed and the species of nucleotide may be changed during repeated cycles of synthesis. 1. A method for enzymatic synthesis of polynucleotides , the method comprising:a) attaching acyl groups to 3′ ends of a plurality of initiators attached to a microelectrode array;b) incubating the microelectrode array in a deacylation solution;c) activating a subset of electrodes in the microelectrode array to generate a negative voltage at a selected location;d) incubating the microelectrode array with a single species of nucleotide and template independent polymerase; ande) contacting the array with a wash solution.2. A method for enzymatic synthesis of polynucleotides , the method comprising:on an array having a plurality of initiators attached thereto,a) selectively removing 3′ blocking groups from the initiators at a selected location on the array;b) incubating the array with a single species of nucleotide and template-independent polymerase; andc) contacting the array with a wash solution.3. The method of claim 2 , further comprising attaching the 3′ blocking groups to ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of eliciting an immune response in a patient who has cancer , comprising administering to said patient a population of activated T cells that selectively recognize cells that aberrantly present a peptide consisting of the amino acid sequence SEQ ID NO: 32 ,wherein said cancer is selected from chronic lymphocytic leukemia (CLL), ovarian cancer (OC), non-small cell lung cancer (NSCLC), uterine cancer (UEC), and melanoma (MEL).2. The method of claim 1 , wherein the T cells are autologous to the patient.3. The method of claim 1 , wherein the T cells are obtained from a healthy donor.4. The method of claim 1 , wherein the T cells are obtained from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.5. The method of claim 1 , wherein the activated T cells are expanded in vitro.6. The method of claim 1 , wherein the population of activated T cells are administered in the form of a composition.7. The method of claim 6 , wherein the composition further comprises an adjuvant.8. The method of claim 7 , wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 7 , imiquimod claim 7 , resiquimod claim 7 , GM-CSF claim 7 , cyclophosphamide claim 7 , sunitinib claim 7 , bevacizumab claim 7 , interferon-alpha claim 7 , ...

MODIFIED TEMPLATE-INDEPENDENT ENZYMES FOR POLYDEOXYNUCLEOTIDE SYNTHESIS

The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences. 1. A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in the GGFRR or TGSR motifs and capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.2. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-138 bovine TdT and a protein tag sequence fused to the N-terminus.3. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-151 bovine TdT and a protein tag sequence fused to the N-terminus.4. The modified TdT of claim 1 , wherein the modified TdT comprises an N-terminus t-160 bovine TdT and a protein tag sequence fused to the N-terminus.5. The modified TdT of claim 1 , wherein the modified TdT exhibits an increased rate of incorporation of modified nucleotides claim 1 , compared to native TdT.6. The modified TdT of claim 1 , wherein the modified TdT is capable of adding adenine claim 1 , cytosine claim 1 , guanine claim 1 , and thymine deoxyribonucleotides modified with a removable 3′-O-blocking moiety.7. The modified TdT of claim 6 , wherein said nucleotides are 2′-deoxyribonucleotides.8. The modified TdT of claim 1 , wherein the modified TdT is capable of adding adenine claim 1 , cytosine claim 1 , guanine claim 1 , and uracil ribonucleotides modified with a removable 3′-O-blocking moiety.9. The modified TdT of claim 1 , wherein said removable 3′-O-blocking moiety comprises a 3′-O-azidomethyl group.10. The modified TdT of claim 1 , wherein said removable 3′-O-blocking ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide consisting of the amino acid sequence ALNPELVQA (SEQ ID NO: 62) in the form of a pharmaceutically acceptable salt.2. The peptide of claim 1 , wherein said peptide has the ability to bind to an MHC class-I molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD8 T cells.3. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is chloride salt.4. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is acetate salt.5. A composition comprising the peptide of claim 1 , wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.6. The composition of claim 5 , wherein the peptide is in the form of a chloride salt.7. The composition of claim 5 , wherein the peptide is in the form of an acetate salt.8. The composition of wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 5 , imiquimod claim 5 , resiquimod claim 5 , GM-CSF claim 5 , cyclophosphamide claim 5 , sunitinib claim 5 , bevacizumab claim 5 , interferon-alpha claim 5 , interferon-beta claim 5 , CpG oligonucleotides and derivatives claim 5 , poly-(I:C) and derivatives claim 5 , RNA claim 5 , sildenafil claim 5 , particulate formulations ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide consisting of the amino acid sequence FINDSIVYL (SEQ ID NO: 106) in the form of a pharmaceutically acceptable salt.2. The peptide of claim 1 , wherein said peptide has the ability to bind to an MHC class-I molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD8 T cells.3. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is chloride salt.4. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is acetate salt.5. A composition comprising the peptide of claim 1 , wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.6. The composition of claim 5 , wherein the peptide is in the form of a chloride salt.7. The composition of claim 5 , wherein the peptide is in the form of an acetate salt.8. The composition of wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 5 , imiquimod claim 5 , resiquimod claim 5 , GM-CSF claim 5 , cyclophosphamide claim 5 , sunitinib claim 5 , bevacizumab claim 5 , interferon-alpha claim 5 , interferon-beta claim 5 , CpG oligonucleotides and derivatives claim 5 , poly-(I:C) and derivatives claim 5 , RNA claim 5 , sildenafil claim 5 , particulate formulations ...

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Use of Terminal Transferase Enzyme in Nucleic Acid Synthesis

The invention relates to the use of a modified terminal transferase enzyme in a method of adding one or more nucleotides to the 3′ end of a nucleic acid. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of said modified terminal transferase enzyme, to kits comprising said modified terminal transferase enzyme and to the use of said kits in methods of nucleic acid synthesis and sequencing. 1. Use of a modified terminal transferase enzyme in a method of adding one or more nucleotides to the 3′ end of a nucleic acid , characterised in that said enzyme comprises a mutated BRCA-1 C-terminal (BRCT) domain.2. The use as defined in claim 1 , wherein the terminal transferase enzyme is from the DNA polymerase X family.3. The use as defined in or claim 1 , wherein the terminal transferase enzyme is selected from terminal deoxynucleotidyl transferase (TdT) claim 1 , DNA polymerase A (Poll) and DNA polymerase μ (Polμ) claim 1 , such as terminal deoxynucleotidyl transferase (TdT).4. The use as defined in any one of to claim 1 , wherein said BRCT domain contains one or more mutations selected from: a deletion claim 1 , substitution or an insertion.5. The use as defined in any one of to claim 1 , wherein said enzyme comprises a truncated BRCT domain claim 1 , such as an N-terminal truncated BRCT domain.6. The use as defined in any one of to claim 1 , wherein said BRCT domain is absent.7. The use as defined in any one of to claim 1 , wherein said method is nucleic acid synthesis.8. A method of nucleic acid synthesis claim 1 , which comprises the steps of:(a) providing an initial initiator sequence;{'claim-ref': [{'@idref': 'CLM-00001', 'claims 1'}, {'@idref': 'CLM-00006', '6'}], '(b) adding a reversibly blocked nucleotide triphosphate to said initiator sequence in the presence of a modified terminal transferase enzyme as defined in any one of to ;'}(c) removal of all reagents from the initiator sequence;(d) cleaving the blocking group ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide consisting of the amino acid sequence WYVNGVNYF (SEQ ID NO: 189) in the form of a pharmaceutically acceptable salt.2. The peptide of claim 1 , wherein said peptide has the ability to bind to an MHC class-I molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD8 T cells.3. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is chloride salt.4. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is acetate salt.5. A composition comprising the peptide of claim 1 , wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.6. The composition of claim 5 , wherein the peptide is in the form of a chloride salt.7. The composition of claim 5 , wherein the peptide is in the form of an acetate salt.8. The composition of wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 5 , imiquimod claim 5 , resiquimod claim 5 , GM-CSF claim 5 , cyclophosphamide claim 5 , sunitinib claim 5 , bevacizumab claim 5 , interferon-alpha claim 5 , interferon-beta claim 5 , CpG oligonucleotides and derivatives claim 5 , poly-(I:C) and derivatives claim 5 , RNA claim 5 , sildenafil claim 5 , particulate formulations ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide consisting of the amino acid sequence ALLGTKILL (SEQ ID NO: 105) in the form of a pharmaceutically acceptable salt.2. The peptide of claim 1 , wherein said peptide has the ability to bind to an MHC class-I molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD8 T cells.3. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is chloride salt.4. The peptide of claim 1 , wherein the pharmaceutically acceptable salt is acetate salt.5. A composition comprising the peptide of claim 1 , wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.6. The composition of claim 5 , wherein the peptide is in the form of a chloride salt.7. The composition of claim 5 , wherein the peptide is in the form of an acetate salt.8. The composition of wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 5 , imiquimod claim 5 , resiquimod claim 5 , GM-CSF claim 5 , cyclophosphamide claim 5 , sunitinib claim 5 , bevacizumab claim 5 , interferon-alpha claim 5 , interferon-beta claim 5 , CpG oligonucleotides and derivatives claim 5 , poly-(I:C) and derivatives claim 5 , RNA claim 5 , sildenafil claim 5 , particulate formulations ...

Single-cell level connectomics using a dna-synthesis based barcoding system and methods of using the same

The present disclosure provides a DNA-synthesis based recording system that, in combination with CRISPR-Cas9 or other CRISPR systems, can establish single-cell level connectivity for densely packed cells, for example in the brain.

DOUBLE-STRAND DNA BREAK QUANTIFICATION METHOD

The present disclosure provides the quantification of double-strand breaks in DNA molecules using terminal deoxynucleotidyl transferase using a preliminary step of nick gap and repair. This preliminary step comprising contacting the DNA molecules with both a DNA ligase and a DNA polymerase to repair DNA nicks and remove DNA gaps prior to using the terminal deoxynucleotidyl transferase. 1. A method for quantifying double-stand breaks in a sample comprising DNA molecules susceptible of having at least one DNA nick , at least one DNA gap and at least one DNA double strand break , the method comprising:(a) contacting the sample with a DNA ligase and a DNA polymerase under conditions to (i) allow the reparation of the at least one DNA nick and the at least one DNA gap, (ii) inhibit the amplification of the DNA molecules and (iii) obtain a first DNA mixture comprising a first DNA molecule having a 3′-OH free terminus;(b) contacting the first DNA mixture with a terminal deoxynucleotidyl transferase (TdT) and a substrate of the TdT, said substrate comprising a nucleotide having a label, under conditions so as to allow the incorporation of the nucleotide at the 3′-OH free terminus of the first DNA molecule to obtain a second DNA mixture comprising second DNA molecules having the incorporated nucleotide;(c) purifying the second DNA molecules from the second DNA mixture to obtain at substantially isolated DNA molecules;(d) optionally amplifying, arraying and/or sequencing the substantially isolated DNA molecules obtained at step (c); and(e) quantifying a signal associated with the substantially isolated DNA molecules obtained at step (c), the amplification of the substantially isolated DNA molecules obtained at step (d), the array of the substantially isolated DNA molecules obtained at step (d) and/or the sequence of the substantially isolated DNA molecules obtained at step (d) to quantify and optionally locate DNA double-strand breaks in the sample.2. The method of claim 1 , ...

Non-human animals expressing exogenous terminal deoxynucleotidyltransferase

Provided herein are methods and compositions related to non-human animals that express exogenous Terminal Deoxynucleotidyltransferase (TdT).

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide comprising an amino acid sequence comprising SEQ ID No. 305 , and/or a variant sequence thereof which is at least 88% homologous to SEQ ID No. 305 , wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and/or induces T cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof , wherein said peptide is not a full-length polypeptide.2. The peptide or variant according to claim 1 , wherein said peptide has the ability to bind to a MHC class-I or -II molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD4 and/or CD8 T cells.3. The peptide or variant thereof according to claim 1 , wherein the amino acid sequence thereof comprises a continuous stretch of amino acids according to the group of SEQ ID No. 305.4. The peptide or variant thereof according to claim 1 , wherein said peptide or variant thereof has an overall length of from 8 to 100 claim 1 , optionally from 8 to 30 claim 1 , and optionally from 8 to 16 amino acids claim 1 , and optionally wherein the peptide consists or consists essentially of an amino acid of SEQ ID No. 305.5. The peptide or variant thereof according to claim 1 , wherein said peptide is part ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide comprising an amino acid sequence comprising SEQ ID No. 306 , and/or a variant sequence thereof which is at least 88% homologous to SEQ ID No. 306 , wherein said variant binds to molecule(s) of the major histocompatibility complex (MHC) and/or induces T cells cross-reacting with said variant peptide; and a pharmaceutical acceptable salt thereof , wherein said peptide is not a full-length polypeptide.2. The peptide or variant according to claim 1 , wherein said peptide has the ability to bind to a MHC class-I or -II molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD4 and/or CD8 T cells.3. The peptide or variant thereof according to claim 1 , wherein the amino acid sequence thereof comprises a continuous stretch of amino acids according to the group of SEQ ID No. 306.4. The peptide or variant thereof according to claim 1 , wherein said peptide or variant thereof has an overall length of from 8 to 100 claim 1 , optionally from 8 to 30 claim 1 , and optionally from 8 to 16 amino acids claim 1 , and optionally wherein the peptide consists or consists essentially of an amino acid of SEQ ID No. 306.5. The peptide or variant thereof according to claim 1 , wherein said peptide is part ...

MODIFIED TEMPLATE-INDEPENDENT ENZYMES FOR POLYDEOXYNUCLEOTIDE SYNTHESIS

The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences. 1. A modified terminal deoxynucleotidyl transferase (TdT) comprising one or more mutations selected from the group consisting of E180D and W450H , M192E , E180K , E180K and R454A , R454K , M192K , M192K and E180K , Q455I , M192W , E180R , E180L , M192R , N474R , R461V , E180K and R454I , E180D and M192E , E180D and M192E and R454T , and E180K and W450H , said modified TdT capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.2. The modified TdT of claim 1 , wherein the modified TdT comprises N454R.3. The modified TdT of claim 1 , wherein the modified TdT comprises R461V4. The modified TdT of claim 1 , wherein the modified TdT comprises R454K.5. The modified TdT of claim 1 , wherein the modified TdT comprises E180K and R454A.6. The modified TdT of claim 1 , wherein the modified TdT comprises M192K and E180K.7. The modified TdT of claim 1 , wherein the modified TdT comprises E180K.8. The modified TdT of claim 1 , wherein the modified TdT comprises M192K.9. The modified TdT of claim 1 , wherein the modified TdT comprises E180L.10. The modified TdT of claim 1 , wherein the modified TdT comprises E180D and W450H.11. The modified TdT of claim 1 , wherein the modified TdT comprises M192E.12. The modified TdT of claim 1 , wherein the modified TdT comprises Q455I.13. The modified TdT of claim 1 , wherein the modified TdT comprises E180K and W450H.14. The modified TdT of claim 1 , wherein the modified TdT comprises M192W.15. The modified TdT of claim ...

Modified template-independent enzymes for polydeoxynucleotide synthesis

The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.

Enzymatic DNA Synthesis Using the Terminal Transferase Activity of Template-Dependent DNA Polymerases

Methods for making a polynucleotide is provided. The methods include (a) providing a first single stranded oligonucleotide, (b) providing a second single stranded oligonucleotide under conditions wherein the first single stranded oligonucleotide anneals to the second single stranded oligonucleotide thereby forming a double stranded oligonucleotide template having an extendible end comprising the 3′ terminal nucleotide of the first single stranded oligonucleotide, (c) providing a reaction mixture to the double stranded initiator wherein the reaction mixture comprises an enzyme, a selected nucleotide triphosphate, and divalent cations, and wherein the enzyme extends the extendible end, (d) regenerating an extendible end of the extended template, and repeating steps (c) to (d) until a polynucleotide of a desired sequence or information content is formed, with the proviso that step (d) is not required to be performed after the polynucleotide is formed. 1. A method for adding one or more selected nucleotides to an extendible end of a double stranded oligonucleotide initiator comprising(a) providing a first single stranded oligonucleotide(b) providing a second single stranded oligonucleotide under conditions wherein the first single stranded oligonucleotide anneals to the second single stranded oligonucleotide thereby forming the double stranded oligonucleotide initiator having an extendible end comprising a 3′ terminal nucleotide of the first single stranded oligonucleotide,(c) providing a reaction mixture to the double stranded initiator wherein the reaction mixture comprises a template-dependent DNA polymerase, one or more selected nucleotide triphosphates, and divalent cations, and wherein the template-dependent DNA polymerase adds one or more of the selected nucleotide triphosphates to the 3′ terminal nucleotide of the first single stranded oligonucleotide of the extendible end of the double stranded oligonucleotide initiator.2. The method of wherein 3′ end terminal ...

Azidomethyl Ether Deprotection Method

The invention relates to a method of converting an azidomethyl ether substituent to a free hydroxyl group. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of nucleotide triphosphates having a 3′-O-azidomethyl substituent, to kits comprising nucleotide triphosphates having a 3′-O-azidomethyl substituent and photoactivatable transition metal complex and to the use of said kits in methods of nucleic acid synthesis and sequencing. 1. A method of converting an azidomethyl ether substituent to a free hydroxyl group wherein said method comprises the step of exposing a compound having said azidomethyl ether substituent to a photoactivated transition metal complex.2. The method as defined in claim 1 , wherein the photoactivated transition metal complex comprises a transition metal selected from ruthenium claim 1 , platinum claim 1 , palladium claim 1 , rhodium and osmium.3. The method as defined in claim 2 , wherein the transition metal is ruthenium.4. The method as defined in any one of to claim 2 , wherein the photoactivated transition metal complex comprises a ligand which is a mono-dentate or bidentate ligand selected from phosphine claim 2 , thiocynate claim 2 , nitrogen claim 2 , pyridine claim 2 , phenanthroline claim 2 , cyclopentadienyl and N-heterocyclic carbine based ligands.5. The method as defined in claim 4 , wherein the photoactivated transition metal complex comprises a pyridine ligand claim 4 , such as a bipyridine ligand.6. The method as defined in claim 5 , wherein the photoactivated transition metal complex is tris(2 claim 5 ,2′-bipyridyl)ruthenium(II)).7. The method as defined in any one of to claim 5 , wherein the azidomethyl ether is present on a ribose or deoxyribose sugar moiety.8. The method as defined in claim 7 , wherein the azidomethyl ether is a 2′ or 3′-O-azidomethyl.9. The method as defined in any one of to claim 7 , which comprises the step of exposing a compound having said azidomethyl ether ...

ELECTROCHEMICALLY-CLEAVABLE LINKERS

This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis. 2. The compound of claim 1 , wherein P is present claim 1 , Y is present claim 1 , Lis present claim 1 , Cis present claim 1 , Lis omitted claim 1 , and Lis present.3. The compound of claim 1 , wherein P is present and a peptide claim 1 , wherein the peptide is an enzyme.4. The compound of claim 3 , wherein the enzyme is TdT.5. The compound of claim 1 , wherein P is present and a linked nucleotide comprising at least one of DNA claim 1 , RNA claim 1 , or a synthetic nucleotide having a universal base.6. The compound of claim 1 , wherein P is present and a linked nucleotide that is complementary to the nucleotide.8. The compound of claim 1 , wherein Lis omitted.11. The compound of claim 10 , wherein Xis hydrogen and Xis hydrogen.12. The compound of claim 10 , wherein Xis a methyl ether and Xis hydrogen.17. The compound of claim 1 , wherein the nucleotide comprises a DNA nucleotide triphosphate or an RNA nucleotide triphosphate.18. The compound of claim 1 , wherein the base of the nucleotide is a pyrimidine base and Lis attached to the number 5 carbon of the pyrimidine base or the base of the nucleotide is a purine base ...

Variants of Terminal Deoxynucleotidyl Transferase and Uses Thereof

The present invention relates to a variant of Terminal deoxynucleotidyl Transferase (TdT) which (i) comprises the amino acid sequence as set forth in SEQ ID No 2 or a functionally equivalent sequence, with at least an amino acid substitution at position corresponding to residue C302 or functionally equivalent residue, wherein the position is numbered by reference to the amino acid sequence set forth in SEQ ID No 1, (ii) is able to synthesize a nucleic acid fragment without template and (iii) is able to incorporate a modified nucleotide into the nucleic fragment.

COMPOSITIONS AND METHODS RELATED TO NUCLEIC ACID PREPARATION

The invention relates to a method of nucleic acid synthesis comprising the use of 3′-O-azidomethyl blocked nucleotide triphosphates which comprises the step of adding a capping group to any uncleaved 3′-O-azidomethyl groups and to the use of kits comprising said capping groups in a method of nucleic acid synthesis. The invention also relates to capped nucleotide triphosphates and 3′-O-azidomethyl capping groups. 1. A method of treating an oligonucleotide , which comprises the steps of:(a) providing an oligonucleotide with a 3′-O-azidomethyl group an initiator sequence; and(b) treating the 3′-O-azidomethyl group via a 1,3-dipolar cycloaddition reaction to prevent subsequent cleavage of the 3′-O-azidomethyl group.2. The method as defined in claim 1 , wherein the 3′-O-azidomethyl group is treated with an irreversible capping group.3. The method as defined in claim 2 , wherein the capping group is a dipolarophile.4. The method as defined in claim 3 , wherein the dipolarophile is an alkyne claim 3 , such as a strained alkyne.5. The method as defined in claim 3 , wherein the dipolarophile is dibenzocyclooctyne-amine.6. The method as defined in claim 1 , wherein the 1 claim 1 ,3-dipolar cycloaddition reaction of step (b) comprises an uncatalysed cycloaddition reaction.7. The method as defined in claim 1 , wherein the 1 claim 1 ,3-dipolar cycloaddition reaction of step (b) comprises a cycloaddition reaction catalysed by a copper or ruthenium-based catalyst.8. The method as defined in claim 2 , wherein the capping group comprises biotin.9. The method as defined in claim 2 , wherein the capping group comprises a fluorine containing moiety.10. The method as defined in claim 2 , wherein the capping group comprises a fluorescent moiety.1216-. (canceled)17. The method as defined in claim 1 , wherein the capping group is an alkyne containing reagent.20. (canceled) This application is a continuation of U.S. patent application Ser. No. 15/555,232 filed Sep. 1, 2017, which is a ...

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

METHODS AND APPARATUS FOR SYNTHESIZING NUCLEIC ACIDS

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems. 1. A method for non-template dependent oligonucleotide synthesis , the method comprising:exposing a nucleic acid strand to a terminal transferase enzyme capable of incorporating a single nucleotide and remaining bound to the strand and preventing further nucleotide incorporation until exposed to a releasing agent or releasing condition.2. The method of claim 1 , wherein said single nucleotide is a nucleotide analog.3. The method of claim 1 , wherein the terminal transferase enzyme is a modified terminal deoxynucleotidyl transferase (TdT) enzyme.4. The method of claim 3 , wherein the modification comprises a mutation allowing the covalent attachment of a nucleotide analog to the TdT enzyme.5. The method of claim 1 , wherein the releasing reagent comprises a salt buffer or a denaturant or a reducing agent or elevated pH.6. The method of claim 1 , wherein the releasing condition is a temperature increase or agitation.7. A method for non-template dependent oligonucleotide synthesis claim 1 , the method comprising:exposing a covalent blocker-nucleic acid strand complex to an exonuclease to remove un complexed, unmodified nucleic acid strands;removing the exonuclease; andexposing the covalent blocker-nucleic acid strand complex to a releasing agent or a releasing condition. This Application is a continuation of U.S. patent application Ser. No. 16/026,662, filed Jul. 3, 2018, which is a continuation of U.S. patent application ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of eliciting an immune response in a patient who has cancer , comprising administering to said patient a population of activated T cells that selectively recognize cells that aberrantly present a peptide consisting of the amino acid sequence selected from SEQ ID NO: 27 , SEQ ID NO: 1 , SEQ ID NO: 3 to SEQ ID NO: 23 , SEQ ID NO: 25 to SEQ ID NO: 31 , SEQ ID NO: 33 to SEQ ID NO: 38 , SEQ ID NO: 40 to SEQ ID NO: 63 , SEQ ID NO: 65 to SEQ ID NO: 71 , SEQ ID NO: 73 to SEQ ID NO: 104 , SEQ ID NO: 107 to SEQ ID NO: 148 , SEQ ID NO: 150 to SEQ ID NO: 153 , SEQ ID NO: 155 to SEQ ID NO: 188 , SEQ ID NO: 190 to SEQ ID NO: 219 , SEQ ID NO: 221 to SEQ ID NO: 250 , SEQ ID NO: 252 to SEQ ID NO: 270 , and SEQ ID NO: 272 to SEQ ID NO: 302 ,wherein said cancer is selected from glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, Non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gallbladder cancer, cholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer, head and neck squamous cell carcinoma, and uterine cancer.2. The method of claim 1 , wherein the T cells are ...

MODIFIED TEMPLATE-INDEPENDENT DNA POLYMERASE

Described herein are genetically engineered template-independent DNA polymerases, specifically terminal deoxynucleotidyl transferases, and methods of using these polymerases to control DNA synthesis by adding a single nucleotide (mononucleotide) at a time to the 3′ end of a growing single-stranded DNA polynucleotide. 1. An engineered terminal deoxynucleotidyl transferase (TdT) , wherein one , or more , amino acid residues of the TdT are modified , resulting in a TdT capable of controlled addition of nucleotides to the 3′ end of a single-stranded polynucleotide.2. The TdT of claim 1 , wherein the TdT comprises the amino acid sequence SEQ ID NO:1 (murine amino acid sequence) claim 1 , SEQ ID NO:5 claim 1 , or a homologous TdT comprising at least about 50% sequence identity with SEQ ID NO:1 or SEQ ID NO:5.3. The TdT of claim 1 , wherein the engineered TdT is photoisomerizable.4. The TdT of claim 3 , wherein one claim 3 , or more amino acid residues of the TdT are substituted with a non-naturally occurring amino acid comprising a reactive group that can be chemically crosslinked.5. The TdT of wherein the chemically reactive amino acid is crosslinked to a photoswitchable moiety.6. The TdT of claim 5 , wherein the photoswitchable moiety is an azobenzene derivative.7. The TdT of claim 6 , wherein the azobenzene derivative regulates/gates entry or binding of a mononucleotide to the active site of TdT.8. The TdT of claim 6 , wherein the azobenzene derivative is modified by a chemically reactive group.9. The TdT of claim 4 , wherein the substituted amino acid residues are residues exposed on the surface of the TdT protein or are residues that do not interfere with the enzymatic activity of the TdT protein.10. The TdT of wherein the amino acid residues are lysine residues.11. The TdT of claim 10 , wherein the lysine residues are selected from residues at positions 199 claim 10 , 238 claim 10 , 247 claim 10 , 250 claim 10 , 276 claim 10 , 338 or 419 of the amino acid sequence ...

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of eliciting an immune response in a patient who has cancer , comprising administering to said patient a population of activated T cells that selectively recognize cells that aberrantly present a peptide consisting of the amino acid sequence of ALLGTKILL (SEQ ID NO: 105) ,wherein said cancer is selected from the group consisting of non-Hodgkin lymphoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal cancer, lower grade glioma, ovarian cancer, skin cutaneous melanoma, stomach adenocarcinoma, chronic lymphocytic leukemia, glioblastoma, small cell lung cancer, and urinary bladder cancer.2. The method of claim 1 , wherein the T cells are autologous to the patient.3. The method of claim 1 , wherein the T cells are obtained from a healthy donor.4. The method of claim 1 , wherein the T cells are obtained from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.5. The method of claim 1 , wherein the activated T cells are expanded in vitro.6. The method of claim 1 , wherein the population of activated T cells are administered in the form of a composition.7. The method of claim 6 , wherein the composition further comprises an adjuvant.8. The method of claim 7 , wherein the adjuvant is selected from the group consisting of anti- ...

Reusable initiators for synthesizing nucleic acids

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

METHODS AND APPARATUS FOR SYNTHESIZING NUCLEIC ACIDS

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems. 1. A method for non-template dependent oligonucleotide synthesis , the method comprising:exposing a nucleic acid strand to a terminal transferase enzyme capable of incorporating a single nucleotide and remaining bound to the strand and preventing further nucleotide incorporation until exposed to a releasing agent or releasing condition.2. The method of claim 1 , wherein said single nucleotide is a nucleotide analog.3. The method of claim 1 , wherein the terminal transferase enzyme is a modified terminal deoxynucleotidyl transferase (TdT) enzyme.4. The method of claim 3 , wherein the modification comprises a mutation allowing the covalent attachment of a nucleotide analog to the TdT enzyme.5. The method of claim 1 , wherein the releasing reagent comprises a salt buffer or a denaturant or a reducing agent or elevated pH.6. The method of claim 1 , wherein the releasing condition is a temperature increase or agitation.7. A nucleotidyl transferase enzyme modified to remain bound to a nucleic acid strand after incorporating a nucleotide into the nucleic acid strand and to prevent subsequent incorporation of nucleotide analogs until released by a releasing agent or releasing condition.8. The nucleotidyl transferase enzyme of claim 7 , wherein the nucleotidyl transferase enzyme incorporates the nucleotide analog into the nucleic acid strand in an absence of a nucleic acid template.9. The nucleotidyl transferase enzyme of claim 7 ...

Compositions and Methods Related to Nucleic Acid Synthesis

The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis. 138-. (canceled)39. A method of nucleic acid synthesis , which comprises:(a) providing an initiator oligonucleotide immobilised on a solid support; and(b) adding a 3′-blocked nucleoside triphosphate to said initiator in the presence of a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 1 to 5 and 8 or a fragment of at least 300 amino acids thereof.40. The method as defined in claim 39 , further comprising the steps of:(c) removal of all reagents from the initiator oligonucleotide;(d) cleaving the blocking group from the 3′-blocked nucleoside in the presence of a cleaving agent; and(e) removal of the cleaving agent.41. The method as defined in claim 39 , wherein the terminal deoxynucleotidyl transferase (TdT) comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a fragment of at least 300 amino acids thereof.42. The method as defined in claim 40 , wherein greater than 1 nucleotide is added by repeating steps (b) to (e).43. The method as defined in claim 39 , wherein the 3′-blocked nucleoside triphosphate is blocked by either a 3′-O-azidomethyl claim 39 , 3′-aminoxy or 3′-O-allyl group.44Saccharomyces cerevisiae.. The method as defined in claim 39 , wherein the terminal deoxynucleotidyl transferase (TdT) is added in the presence of an extension solution comprising one or more buffers claim 39 , such as Tris or cacodylate claim 39 , one or more salts claim 39 , and inorganic pyrophosphatase claim 39 , such ...

Non-Human Animals Expressing Exogenous Terminal Deoxynucleotidyltransferase

Provided herein are methods and compositions related to non-human animals that express exogenous Terminal Deoxynucleotidyltransferase (TdT). 1. A genetically modified non-human animal comprising in its genome:a nucleic acid sequence encoding an exogenous Terminal Deoxynucleotidyltransferase (TdT) operably linked to a transcriptional control element; andan immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments operably linked to an immunoglobulin constant region gene.2. The genetically modified non-human animal of claim 1 , wherein the transcriptional control element drives expression of the nucleic acid sequence encoding the exogenous TdT in pro-B cells and/or pre-B cells.3. The genetically modified non-human animal of claim 2 , wherein the transcriptional control element is selected from the group consisting of a RAG1 transcriptional control element claim 2 , a RAG2 transcriptional control element claim 2 , an immunoglobulin heavy chain transcriptional control element claim 2 , an immunoglobulin κ light chain transcriptional control element and/or an immunoglobulin λ light chain transcriptional control element.4. The genetically modified non-human animal of any one of claims 2 , wherein the nucleic acid sequence encoding the exogenous TdT is located at an immunoglobulin κ light chain locus claims 2 , an immunoglobulin λ light chain locus claims 2 , an immunoglobulin heavy chain locus claims 2 , a RAG1 locus claims 2 , a RAG2 locus.5. The genetically modified non-human animal of claim 1 , wherein at least 10% of the V-J immunoglobulin light chain junctions in the animal comprise non-template additions.6. The genetically modified non-human animal of claim 1 , wherein the human immunoglobulin variable region gene segments are human heavy chain variable region gene segments.7. The genetically modified non-human animal of claim 6 , wherein the constant region gene is a heavy chain constant region gene.8. The genetically ...

Genome editing systems comprising repair-modulating enzyme molecules and methods of their use

The application provides improved methods of genome editing. The genome editing systems described herein comprise a RNA-guided nuclease molecule and a Repair-Modulating Enzyme Molecule (RMEM).

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A method of eliciting an immune response in a patient who has cancer , comprising administering to said patient a population of activated T cells that selectively recognize cells , which present a peptide consisting of the amino acid sequence GYIDNVTLI (SEQ ID NO: 220) ,wherein the peptide is in a complex with an MHC molecule,wherein said cancer is selected from the group consisting of glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, chronic lymphocytic leukemia, hepatocellular carcinoma, non-small cell and small cell lung cancer, non-Hodgkin lymphoma, acute myeloid leukemia, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer including cancer of the gastric-esophageal junction, gallbladder cancer and cholangiocarcinoma, melanoma, gastric cancer, urinary bladder cancer, head-and neck squamous cell carcinoma, and uterine cancer.2. The method of claim 1 , wherein the T cells are autologous to the patient.3. The method of claim 1 , wherein the T cells are obtained from a healthy donor.4. The method of claim 1 , wherein the T cells are obtained from tumor infiltrating lymphocytes or peripheral blood mononuclear cells.5. The method of claim 1 , wherein the activated T cells are expanded in vitro.6. The method of claim 1 , ...

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Processive Template Independent DNA Polymerase Variants

An enzymatic method of making a polynucleotide is provided. The method includes combining a selected nucleotide triphosphate, one or more cations, a template-independent polymerase, and an associated processivity factor in an aqueous reaction medium including a target substrate comprising an initiator sequence and having a 3′ terminal nucleotide attached to a single stranded portion, such that the template-independent polymerase and the associated processivity factor interact with the target substrate under conditions which covalently add one or more of the selected nucleotide triphosphate to the 3′ terminal nucleotide. Also provided are mutant template-independent polymerases having a processivity factor attached thereto. 1. An enzymatic method of making a polynucleotide comprisingcombining a selected nucleotide triphosphate, one or more cations, a template-independent polymerase, and an associated processivity factor in an aqueous reaction medium including a target substrate comprising an initiator sequence and having a 3′ terminal nucleotide attached to a single stranded portion, such that the template-independent polymerase and the associated processivity factor interact with the target substrate under conditions which covalently add one or more of the selected nucleotide triphosphate to the 3′ terminal nucleotide.2. The method of further includingrepeatedly introducing a subsequent selected nucleotide triphosphate to the aqueous reaction medium under conditions which enzymatically add one or more of the subsequent selected nucleotide triphosphate to the target substrate until the polynucleotide is formed.3. The method of wherein the processivity factor increases processivity of the template-independent polymerase.4. The method of wherein the processivity factor comprises one or more binding units.5. The method of wherein the processivity factor binds to and translocates across the target substrate.6. The method of wherein the processivity factor binds to and ...

Variants of Terminal Deoxynucleotidyl Transferase and Uses Thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

NOVEL PEPTIDES AND COMBINATION OF PEPTIDES FOR USE IN IMMUNOTHERAPY AGAINST VARIOUS CANCERS

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules. 1. A peptide consisting of the amino acid sequence ALSVLRLAL (SEQ ID NO: 251) in the form of a pharmaceutically acceptable salt.2. The peptide of claim 1 , wherein said peptide has the ability to bind to an MHC class-I molecule claim 1 , and wherein said peptide claim 1 , when bound to said MHC claim 1 , is capable of being recognized by CD8 T cells.3. The peptide of claim 1 , wherein the pharmaceutically acceptable is chloride salt.4. The peptide of claim 1 , wherein the pharmaceutically acceptable is acetate salt.5. A composition comprising the peptide of claim 1 , wherein the composition comprises an adjuvant and a pharmaceutically acceptable carrier.6. The composition of claim 5 , wherein the peptide is in the form of a chloride salt.7. The composition of claim 5 , wherein the peptide is in the form of an acetate salt.8. The composition of wherein the adjuvant is selected from the group consisting of anti-CD40 antibody claim 5 , imiquimod claim 5 , resiquimod claim 5 , GM-CSF claim 5 , cyclophosphamide claim 5 , sunitinib claim 5 , bevacizumab claim 5 , interferon-alpha claim 5 , interferon-beta claim 5 , CpG oligonucleotides and derivatives claim 5 , poly-(I:C) and derivatives claim 5 , RNA claim 5 , sildenafil claim 5 , particulate formulations with poly( ...

Variants of Terminal Deoxynucleotidyl Transferase and Uses Thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment. 1144.-. (canceled)145. A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least 90% identical to SEQ ID N0:2 , SEQ ID NO:11 , SEQ ID N0:13 , SEQ ID N0:17 , SEQ ID N0:19 , SEQ ID N0:21 , SEQ ID N0:29 , or SEQ ID N0:31 with an amino acid substitution of cysteine at position 173 , wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a 3′-O-modified nucleotide into the nucleic acid fragment , andwherein the TdT variant incorporates the 3′-O-modified nucleotide at a rate greater than that of a wild type TdT.146. The TdT variant of claim 145 , wherein the 3′-O-modified nucleotide is a 3′-O—NH2-nucleoside triphosphate claim 145 , a 3′-O-azidomethyl-nucleo side triphosphate claim 145 , a 3′-O-allyl-nucleoside triphosphate claim 145 , a 3′O-(2-nitrobenzyl)-nucleoside triphosphate claim 145 , or a 3′-O-propargyl-nucleoside triphosphate.147. The TdT variant of claim 145 , wherein the modified nucleotide is incorporated onto a free 3′-hydroxyl of a nucleic acid fragment.148. The TdT variant of claim 145 , further comprising a substitution of methionine at position 63 with respect to SEQ ID NO:2 claim 145 , SEQ ID NO:11 claim 145 , SEQ ID NO:13 claim 145 , SEQ ID NO:17 claim 145 , SEQ ID NO:19 claim 145 , or SEQ ID NO:29.149. The TdT variant of claim 145 , further comprising a substitution of arginine at position 207 with respect to SEQ ID NO:2 claim 145 , SEQ ID NO:11 claim 145 , SEQ ID NO:13 claim 145 , SEQ ID NO:17 claim 145 , SEQ ID NO ...

Increasing Long-Sequence Yields In Template-Free Enzymatic Synthesis of Polynucleotides

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides using chain elongation conditions that suppress the formation of DNA secondary structures including, but not limited to, intra-strand and between-strand duplexes, G-quadruplexes, and the like. In some embodiments, such chain elongation conditions include using 3′-O-blocked dNTP monomers that base protection groups or base analogs that suppress the formation of hydrogen bonding in the polynucleotide being synthesized. 1. A method of synthesizing a polynucleotide having a predetermined sequence , the method comprising the steps of:a) providing an initiator having a free 3′-hydroxyl; andb) repeating until the polynucleotide is synthesized cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked, base protected nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is formed, wherein the elongation conditions are selected to prevent hydrogen bonding or base stacking.2. The method of claim 1 , wherein said elongation conditions provide that at least one 3′-O-blocked nucleoside triphosphate has a base protecting moiety attached to its base to prevent hydrogen bonding.3. The method of claim 2 , wherein said 3′-O-blocked nucleoside triphosphate has a base protecting moiety attached to a nitrogen or to an oxygen of its base.4. The method of claim 3 , wherein said 3′-O-blocked nucleoside triphosphate has said base protecting moiety attached to a nitrogen.5. The method of claim 4 , wherein said nitrogen of said base of said 3′-O-blocked nucleoside triphosphate is an exocyclic nitrogen.6. The method of ...

High Efficiency Template-Free Enzymatic Synthesis of Polynucleotides

The invention is directed to compositions and methods for enzymatic template-free synthesis of polynucleotides wherein different terminal deoxynucleotidyl transferase (TdT) variants are used to incorporate different 3′-O-reversibly blocked deoxynucleoside triphosphates (dNTPs) into a growing chain. In part, the invention is a recognition and appreciation that different TdT variants can be engineered to preferentially incorporate specific dNTPs with higher efficiency than a single “general purpose” TdT variant used to incorporate all four 3-O-reversibly blocked dNTPs. 1. A method of synthesizing a polynucleotide having a predetermined sequence , the method comprising the steps of:a) providing an initiator having a free 3′-hydroxyl; andb) repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a terminal deoxynucleotidyltransferase (TdT) variant so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is synthesized, wherein a first TdT variant elongates the initiator or elongated fragments with a 3′-O-blocked nucleoside triphosphate selected from a first set of 3′-O-blocked nucleoside triphosphates and a second TdT variant, different from the first TdT variant, elongates the initiator or elongated fragments with a 3′-O-blocked nucleoside triphosphate selected from a second set of 3′-O-blocked nucleoside triphosphates, and wherein the first TdT variant elongates the initiator or elongated fragments with 3′-O-blocked nucleoside triphosphates from the first set with greater efficiency than the second TdT variant and the second TdT variant elongates the initiator or elongated fragments with 3′-O-blocked nucleoside triphosphates from ...

Terminal deoxyribonucleoside transferase variant and application thereof

本发明提供一种末端脱氧核糖核苷转移酶变体及其应用，通过在野生型氨基酸序列中特定位点引入突变，提高了其与3’‑OH端修饰的核苷酸的耦合效率，实现了高效可控地用于酶促反应合成核酸分子。本发明还提供一种没有模板链的情况下合成核酸分子的方法。

Generating nucleic acids with modified bases using recombinant terminal deoxynucleotidyl transferase

Disclosed herein include methods of generating a single stranded deoxyribonucleic acid (ssDNA) scaffold comprising nucleotides with modified bases using a recombinant terminal deoxynucleotidyl transferase (TdT). The recombinant TdT can comprise an amino acid sequence that is at least 80% identical to a Bos taurus TdT, or a fragment thereof, and for example comprise one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His420 in the Bos taurus TdT.

genetically modified non-human animal, methods that induce expression of an antibody, t-cell production, a genetically modified non-human and non-human animal, and genetically modified non-human animal cells.

são fornecidos no presente documento métodos e composições relacionados a animais não humanos que expressam desoxinucleotidiltransferase terminal exógena (tdt). Non-human animal-related methods and compositions expressing exogenous terminal deoxynucleotidyltransferase (Tdt) are provided herein.

Non-human animals expressing exogenous terminal deoxynucleotidyl transferase

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, in particular to a genetically modified mouse containing a nucleic acid sequence encoding a human terminal deoxynucleotidyl transferase (TdT), functionally connected to a transcription control element, and a variable area of immunoglobulin, as well as its ES cell. The use of the above-mentioned mouse ES cell for obtaining a genetically modified mouse is also disclosed.EFFECT: invention can be effectively used to induce the expression of an antibody containing a variable human domain.35 cl, 16 dwg, 5 tbl, 10 ex РОССИЙСКАЯ ФЕДЕРАЦИЯ (19) RU (11) (13) 2 753 585 C2 (51) МПК A01K 67/027 (2006.01) C07K 14/705 (2006.01) C12N 15/85 (2006.01) C12N 15/90 (2006.01) C12N 9/12 (2006.01) ФЕДЕРАЛЬНАЯ СЛУЖБА ПО ИНТЕЛЛЕКТУАЛЬНОЙ СОБСТВЕННОСТИ (12) ОПИСАНИЕ ИЗОБРЕТЕНИЯ К ПАТЕНТУ (52) СПК A01K 67/0275 (2021.05); A01K 67/0278 (2021.05); C07K 14/7051 (2021.05); C12N 15/85 (2021.05); C12N 15/907 (2021.05); C12N 9/1264 (2021.05); C12N 5/0606 (2021.05) (21)(22) Заявка: 2018144954, 02.06.2017 02.06.2017 Дата регистрации: 18.08.2021 03.06.2016 US 62/345,524 (43) Дата публикации заявки: 14.07.2020 Бюл. № 20 (45) Опубликовано: 18.08.2021 Бюл. № 23 (86) Заявка PCT: US 2017/035731 (02.06.2017) (87) Публикация заявки PCT: R U 2 7 5 3 5 8 5 WO 2017/210586 (07.12.2017) Адрес для переписки: 119019, Москва, ул. Гоголевский бульвар, 11 (56) Список документов, цитированных в отчете о поиске: US 2015037337 A1, 05.02.2015. LAURENT A. BENTOLILA et al., Constitutive Expression of Terminal Deoxynucleotidyl Transferase in Transgenic Mice Is Sufficient for N Region Diversity to Occur at Any lg locus Throughout B Cell Differentiation, J Immunol, 1997, Vol. 158, pp.715-723. TO-HA THAI et al., Isoforms of Terminal Deoxynucleotidyltransferase: (см. прод.) (54) ЖИВОТНЫЕ, НЕ ЯВЛЯЮЩИЕСЯ ЧЕЛОВЕКОМ, ЭКСПРЕССИРУЮЩИЕ ЭКЗОГЕННУЮ ТЕРМИНАЛЬНУЮ ДЕЗОКСИНУКЛЕОТИДИЛТРАНСФЕРАЗУ (57) Реферат: Изобретение относится к области иммуноглобулина, а также ...

Non-human animals expressing exogenous terminal deoxynucleotidyltransferase

외인성 말단 데옥시뉴클레오타이드 전달효소(TdT)를 발현하는 비인간 동물과 관련된 방법 및 조성물이 본원에 제공된다.

Template-free, high-efficiency enzymatic synthesis of polynucleotides

본 발명은 상이한 3'-O-가역적으로 차단된 데옥시뉴클레오사이드 삼인산(dNTP)을 성장하는 쇄 내로 혼입하기 위해 상이한 말단 데옥시뉴클레오티딜 전달효소(TdT) 변이체를 사용하는 폴리뉴클레오타이드의 무-주형 효소 합성을 위한 조성물 및 방법에 관한 것이다. 부분적으로, 본 발명은 4개의 3'-O-가역적으로 차단된 dNTP를 모두 혼입하기 위해 사용되는 단일한 "범용" TdT 변이체보다 더 높은 효율로 특정 dNTP를 우선적으로 혼입하도록 상이한 TdT 변이체가 조작될 수 있음을 인식하고 이해한다.

Patent RU2018144954A3

ВУ” 2018144954” АЗ Дата публикации: 02.10.2020 Форма № 18 ИЗИМ-2011 Федеральная служба по интеллектуальной собственности Федеральное государственное бюджетное учреждение ж 5 «Федеральный институт промышленной собственности» (ФИПС) ОТЧЕТ О ПОИСКЕ 1. . ИДЕНТИФИКАЦИЯ ЗАЯВКИ Регистрационный номер Дата подачи 2018144954/10(075120) 02.06.2017 РСТ/О$2017/035731 02.06.2017 Приоритет установлен по дате: [ ] подачи заявки [ ] поступления дополнительных материалов от к ранее поданной заявке № [ ] приоритета по первоначальной заявке № из которой данная заявка выделена [ ] подачи первоначальной заявки № из которой данная заявка выделена [ ] подачи ранее поданной заявки № [Х] подачи первой(ых) заявки(ок) в государстве-участнике Парижской конвенции (31) Номер первой(ых) заявки(ок) (32) Дата подачи первой(ых) заявки(ок) (33) Код страны 1. 62/345,524 03.06.2016 05 Название изобретения (полезной модели): [Х] - как заявлено; [ ] - уточненное (см. Примечания) ЖИВОТНЫЕ, НЕ ЯВЛЯЮЩИЕСЯ ЧЕЛОВЕКОМ, ЭКСПРЕССИРУЮЩИЕ ЭКЗОГЕННУЮ ТЕРМИНАЛЬНУЮ ДЕЗОКСИНУКЛЕОТИДИЛТРАНСФЕРАЗУ Заявитель: РЕГЕНЕРОН ФАРМАСЬЮТИКАЛЗ, ИНК., (5 2. ЕДИНСТВО ИЗОБРЕТЕНИЯ [Х] соблюдено [ ] не соблюдено. Пояснения: см. Примечания 3. ФОРМУЛА ИЗОБРЕТЕНИЯ: [Х] приняты во внимание все пункты (см. Примечания) [ ] приняты во внимание следующие пункты: [ ] принята во внимание измененная формула изобретения (см. Примечания) 4. КЛАССИФИКАЦИЯ ОБЪЕКТА ИЗОБРЕТЕНИЯ (ПОЛЕЗНОЙ МОДЕЛИ) (Указываются индексы МПК и индикатор текущей версии) АО1К 67/027 (2006.01) 5. ОБЛАСТЬ ПОИСКА 5.1 Проверенный минимум документации РСТ (указывается индексами МПК) АОТК 67/027 5.2 Другая проверенная документация в той мере, в какой она включена в поисковые подборки: 5.3 Электронные базы данных, использованные при поиске (название базы, и если, возможно, поисковые термины): Е-Габгагу, Езрасепе, РаЗеагсв, РАТЕМТЗСОРЕ, КУРТО, Соозе, боозе Эсво]аг, РиБМеа, О5РТО, 5сепсеПесе 6. ДОКУМЕНТЫ, ОТНОСЯЩИЕСЯ К ПРЕДМЕТУ ПОИСКА Кате- Наименование документа с указанием (где ...

Recombinant bacillus subtilis for synthesizing guanosine diphosphate rock sugar and construction method and application thereof

本发明提供了一种合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用，所述重组芽孢杆菌是通过强化表达枯草芽孢杆菌168的糖转运蛋白基因，并表达外源岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的。本发明通过改造枯草芽孢杆菌168获得了可高效合成合成鸟苷二磷酸岩藻糖的菌株，其胞内积累量高达156mg/L。本发明通过加强表达了糖转运蛋白基因，可以有效强化糖转运蛋白的表达，提高了外源岩藻糖转移至胞内的效率，增加了胞内岩藻糖的浓度，促进了鸟苷二磷酸岩藻糖的合成。本发明的重组枯草芽孢杆菌构建方法简单，便于使用，具有很好的应用前景。

Combinations of novel peptides and peptides for use in immunotherapy for various cancers

본 발명은 면역요법 방법에서의 사용을 위한 펩티드, 단백질, 핵산 및 세포에 관한 것이다. 특히 본 발명은 암의 면역요법에 관한 것이다. 본 발명은 또한 단독 또는 예를 들어 백신 조성물의 활성 약학적 성분으로 역할가능한 기타 종양 연관 펩티드들과 병용하여 항종양 면역 반응을 촉진하거나 또는 세포를 생체외 촉진하여 환자로 이전하기 위한 종양 연관 T 세포 에피톱에 관한 것이다. 주조직적합 복합체(MHC)의 분자에 결합된 펩티드 또한 그러한 펩티드는 항체, 가용성 T 세포 수용체 및 기타 결합하는 분자의 표적일 수도 있다.

POLYMERASE DNA VARIANTS OF THE POLX FAMILY

L'invention concerne des variants d'une ADN polymérase de la famille polX capable de synthétiser une molécule d'acide nucléique sans brin matrice, ou d'un fragment fonctionnel d'une telle polymérase, comprenant au moins une mutation d'un résidu à au moins une position particulière, et des utilisations de ces variants, notamment pour la synthèse de molécules d'acide nucléiques comprenant des nucléotides modifiés en 3'-OH.

Non-human animals expressing exogenous terminal deoxynucleotidyl transferase

本文提供了与表达外源末端脱氧核苷酸转移酶(TdT)的非人动物相关的方法和组合物。

Terminal deoxynucleotidyl transferase variants and uses thereof

The present invention is directed to terminal deoxynucleotidyl transferase (TdT) variants from a variety of species which display enhanced efficiency in incorporating reversibly blocked nucleoside triphosphates into a polynucleotide, and to the use of such TdTs in synthesizing polynucleotides of any predetermined sequence.

Peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor associated T-cell peptide epitopes, alone or in combination with other tumor associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Variants of terminal deoxynucleotidyl transferase and uses thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

Modified template-independent enzymes for polydeoxynucleotide synthesis

本发明包含用于鉴定如经修饰的末端核苷酸基转移酶(TdT)等能够在不使用模板的情况下将包括可移除的3'‑O‑封闭部分的核苷酸与核酸引发剂结合的聚合酶的方法。本发明进一步包含所鉴定的聚合酶以及使用这些聚合酶从头合成预定寡核苷酸序列的方法。

Variants of terminal deoxynucleotidyl transferase and uses thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

Genome editing systems comprising repair-modulating enzyme molecules and methods of their use

The application provides improved methods of genome editing. The genome editing systems described herein comprise a RNA-guided nuclease molecule and a Repair-Modulating Enzyme Molecule (RMEM).

New peptides and peptide combinations for the use of immunotherapy against various types of cancer. (divisional request 201801533)

LA INVENCIÓN SE RELACIONA CON UN PÉPTICO EXPRESADO EN TUMORES, QUE TIENE LA CAPACIDAD DE UNIRSE A UNA MOLÉCULA DEL COMPLEJO PRINCIPAL DE HISTOCOMPATIBILIDAD (MHC) HUMANO DE CLASE I O II, Y EN EL QUE DICHO PÉPTIDO ES CAPAZ DE ESTIMULAR CD4 Y/O CÉLULAS T CD8 ÚTILES EN INMUNOTERAPIA CONTRA EL CÁNCER. THE INVENTION IS RELATED TO A PEPTIC EXPRESSED IN TUMORS, WHICH HAS THE CAPACITY OF BINDING TO A MOLECULE OF THE MAIN HUMAN HISTOCOMPATIBILITY COMPLEX (MHC) OF CLASS I OR II, AND IN WHICH SAID PEPTIDE IS CAPABLE OF STIMULATING CD4 CD8 USEFUL IN IMMUNOTHERAPY AGAINST CANCER.

Nucleotide derivatives containing amine masked moieties and their use in a templated and non-templated enzymatic nucleic acid synthesis

The invention relates to the use of an amine masked moiety in a method of enzymatic nucleic acid synthesis. The invention also relates to said amine masked moieties

Novel use

Chimeric Terminal Deoxynucleotide Transferases for Template-Free Enzymatic Synthesis of Polynucleotides

本発明は、異なる種からのバリアントに由来するキメラである末端デオキシヌクレオチド転移酵素を使用するポリヌクレオチドの酵素的テンプレートフリー合成のための組成物及び方法に関する。【選択図】なし

Use of terminal transferase enzyme in nucleic acid synthesis

The invention relates to the use of a modified terminal transferase enzyme in a method of adding one or more nucleotides to the 3' end of a nucleic acid. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of said modified terminal transferase enzyme, to kits comprising said modified terminal transferase enzyme and to the use of said kits in methods of nucleic acid synthesis and sequencing.

Recombinant terminal deoxynucleotidyl transferase with improved functionality

Truncated terminal deoxynucleotidyl transferase (TdT) derivative from calf thymus, characterized in that the derivative in comparison to the native TdT is N-terminally truncated by up to 161 amino acids and has a 20- to 30-fold higher enzyme activity in solutions containing Co 2+ ions, and its recombinant production and use.

Rescue method of influenza virus and combinations thereof

本发明涉及一种新型流感病毒拯救方法以及其组合物。所述方法包括提供稳定整合并表达流感病毒PA、PB1、PB2和NP基因的宿主细胞，将PA、PB1、PB2和NP基因中分别引入了终止密码子的流感病毒拯救系统导入所述宿主细胞，实现病毒拯救。产生的病毒颗粒可以被用作流感减毒活疫苗，其特点是，由于编码相关蛋白的基因被突变，在人及正常动物细胞中不具有复制增殖能力，仅在上述构建的宿主细胞中可以实现复制增殖，在保证安全性的同时，实现全面激发机体免疫，有效防护机体安全。

A kind of recombined bacillus subtilis for synthesizing guanosine diphosphate fucose and its construction method and application

本发明提供了一种合成鸟苷二磷酸岩藻糖的重组枯草芽孢杆菌及其构建方法与应用，所述重组芽孢杆菌是通过强化表达枯草芽孢杆菌168的糖转运蛋白基因，并表达外源岩藻糖激酶和磷酸鸟嘌呤基转移酶基因得到的。本发明通过改造枯草芽孢杆菌168获得了可高效合成合成鸟苷二磷酸岩藻糖的菌株，其胞内积累量高达156mg/L。本发明通过加强表达了糖转运蛋白基因，可以有效强化糖转运蛋白的表达，提高了外源岩藻糖转移至胞内的效率，增加了胞内岩藻糖的浓度，促进了鸟苷二磷酸岩藻糖的合成。本发明的重组枯草芽孢杆菌构建方法简单，便于使用，具有很好的应用前景。

The variant of the archaeal dna polymerase of polX family

本发明涉及能够在没有模板链的情况下合成核酸分子的polX家族的DNA聚合酶的变体或该聚合酶的功能片段的变体以及所述变体的用途，特别是用于合成包含3'‑OH‑修饰的核苷酸的核酸分子的用途，所述变体包含在至少一个特定位置处的残基的至少一个突变。

Recombinant terminal deoxynucleotidyl transferase with improved functionality

Truncated terminal deoxynucleotidyl transferase (TdT) derivative from calf thymus characterized in that the derivative in comparison to the native TdT is N- terminally truncated by up to 161 amino acids and has a 20- 30-fold higher enzyme activity in solutions containing Co~ions, and it's recombinant production and use.

Chimeric Terminal Deoxynucleotidyl Transferases For Template-Free Enzymatic Synthesis Of Polynucleotides

The invention is directed to compositions and methods for enzymatic template-free synthesis of polynucleotides using terminal deoxynucleotidyl transferases that are chimeras derived from variants from different species.

Template-Free Enzymatic Polynucleotide Synthesis Using Dismutationless Terminal Deoxynucleotidyl Transferase Variants

The present invention is directed to the use of terminal deoxynucleotidyltransferase (TdT) variants lacking dismutation acivity for template-free enzymatic synthesis of polynucleotides of any predetermined sequence. Such TdT variants permit higher yields of correct sequence polynucleotides.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor associated T-cell peptide epitopes, alone or in combination with other tumor associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Base-modified nucleotides as substrates for tdt-based enzymatic nucleic acid

Disclosed herein include methods and compositions for nucleic acid synthesis using a terminal deoxynucleotidyl transferase with deoxyribonucleotide trisphosphates each comprising a modified base with a photocleavable carbon chain moiety that enables single incorporations when present.

Engineered terminal deoxynucleotidyl transferase variants

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis capable of adding a nucleoside triphosphate-3' - O-removable blocking group (NTP-3'-O-RBG) to the 3'-OH end of a growing oligonucleotide or polynucleotide chain in a template-independent manner, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered terminal deoxynucleotidyl transferase.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

Massively parallel enzymatic synthesis of polynucleotides

The invention is directed to methods and compositions for inkjet assisted synthesis of a plurality of polynucleotides at reaction sites on a substrate using template-free polymerases, such as, terminal deoxynucleotidyl transferases (TdTs). Compositions of the invention include formulations of synthesis reagents for inkjet delivery including, but not limited to, TdT coupling reaction buffers and 3'-O-protected dNTP monomers.

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

A kind of terminal deoxy ribonucleotide transfer enzyme variants and its application

本发明提供一种末端脱氧核糖核苷转移酶变体及其应用，通过在野生型氨基酸序列中特定位点引入突变，提高了其与3’‑OH端修饰的核苷酸的耦合效率，实现了高效可控地用于酶促反应合成核酸分子。本发明还提供一种没有模板链的情况下合成核酸分子的方法。

Variants of dna polymerase of polx family

【課題】増強したＤＮＡポリメラーゼを提供する。【解決手段】少なくとも１つの特定の位置における残基の少なくとも１つの変異を含む、鋳型鎖を用いずに核酸分子を合成することができるｐｏｌＸファミリーＤＮＡポリメラーゼまたはそのようなポリメラーゼの機能的断片のバリアント、およびとりわけ３’－ＯＨ修飾ヌクレオチドを含む核酸分子を合成するための上記バリアントの使用に関する。一局面において、鋳型鎖を用いずに核酸分子を合成することができるｐｏｌＸファミリーＤＮＡポリメラーゼまたはそのようなポリメラーゼの機能的断片のバリアントが提供され、上記バリアントは、Ｅ４５７、Ｔ３３１、Ｇ３３２などからなる群より選択される少なくとも１つの位置における残基または機能的に同等な残基の少なくとも１つの変異を含み、示された位置は配列番号１とのアラインメントにより決定される。【選択図】なし

Terminal deoxynucleotidyl transferase variants and uses thereof

本发明涉及来自多种物种的末端脱氧核苷酸转移酶(TdT)变体，其在将可逆性封闭的三磷酸核苷掺入多核苷酸中展示出增强的效率，并且涉及此类TdT在合成任何预定序列的多核苷酸中的用途。

Template-free enzymatic polynucleotide synthesis using dismutationless terminal deoxynucleotidyl transferase variants

The present invention is directed to the use of terminal deoxynucleotidyltransferase (TdT) variants lacking dismutation acivity for template-free enzymatic synthesis of polynucleotides of any predetermined sequence. Such TdT variants permit higher yields of correct sequence polynucleotides.

Compositions and methods for template-free enzymatic nucleic acid synthesis

本揭露提供用於無模板核酸合成的組成物與方法。例示的方法包括去保護包括至少三個核苷酸的引子，其中該引子包括3’可逆的終止核苷酸(rtNTP)；藉由具有終端轉移酶活性的酶或核糖酶，併入至少一游離的rtNTP；並且重複這些步驟，直到產生所欲之合成核酸。可使用在溶液中的引子，及連接至基材之引子(例如，包含陣列)以進行本揭露的方法。

Novel peptides and combination of peptides for use in immunotherapy against various cancers

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

Non-human animals expressing exogenous terminal deoxynucleotidyltransferase.

En la presente se proporcionan métodos y composiciones relacionadas con animales no humanos que expresan desoxinucleotidil transferasa terminal (TdT) exógena.

New peptides and combination of peptides for use in immunotherapy against various types of cancer

LA PRESENTE INVENCIÓN SE REFIERE A PÉPTIDOS, PROTEÍNAS, ÁCIDOS NUCLEICOS Y CÉLULAS DESTINADOS A LA UTILIZACIÓN EN MÉTODOS INMUNOTERAPÉUTICOS. EN PARTICULAR, LA PRESENTE INVENCIÓN SE REFIERE A LA INMUNOTERAPIA CONTRA EL CÁNCER. LA PRESENTE INVENCIÓN SE REFIERE ASIMISMO A EPÍTOPOS PEPTÍDICOS PARA LINFOCITOS T ASOCIADOS A TUMORES, SOLOS O EN COMBINACIÓN CON OTROS PÉPTIDOS ASOCIADOS A TUMORES QUE, POR EJEMPLO, PUEDEN SERVIR COMO PRINCIPIOS ACTIVOS FARMACÉUTICOS EN COMPOSICIONES VACUNALES DESTINADAS A ESTIMULAR RESPUESTAS INMUNITARIAS ANTITUMORALES, O A ESTIMULAR EX VIVO LINFOCITOS T QUE DESPUÉS SERÁN TRANSFERIDOS A LOS PACIENTES. LOS PÉPTIDOS UNIDOS A MOLÉCULAS DEL COMPLEJO MAYOR DE HISTOCOMPATIBILIDAD (MHC), O LOS PÉPTIDOS COMO TALES, TAMBIÉN PUEDEN SER DIANAS DE ANTICUERPOS, DE RECEPTORES DE LINFOCITOS T SOLUBLES, Y DE OTRAS MOLÉCULAS DE UNIÓN. THE PRESENT INVENTION REFERS TO PEPTIDES, PROTEINS, NUCLEIC ACIDS AND CELLS INTENDED FOR USE IN IMMUNOTHERAPEUTIC METHODS. IN PARTICULAR, THE PRESENT INVENTION REFERS TO IMMUNOTHERAPY AGAINST CANCER. THE PRESENT INVENTION ALSO REFERS TO PEPTIDIC EPITOPES FOR T-LYMPHOCYTES ASSOCIATED WITH TUMORS, ALONE OR IN COMBINATION WITH OTHER PEPTIDES ASSOCIATED WITH TUMORS WHICH, FOR EXAMPLE, CAN SERVE AS ACTIVE PRINCIPLES AS AN EXTREME PHARMACEUTICALS ASSOCIATED IN ESTIMATED INCIDENTAL PHARMACEUTICALS, EXPOSED PHARMACEUTICALS LIVE T LYMPHOCYTES WHICH WILL LATER BE TRANSFERRED TO PATIENTS. PEPTIDES ATTACHED TO MOLECULES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC), OR PEPTIDES AS SUCH, MAY ALSO BE TARGETS OF ANTIBODIES, T-SOLUBLE LYMPHOCYTE RECEPTORS, AND OTHER MOLECULES.