WO2012140627A1 - Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer - Google Patents
Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer Download PDFInfo
- Publication number
- WO2012140627A1 WO2012140627A1 PCT/IB2012/051868 IB2012051868W WO2012140627A1 WO 2012140627 A1 WO2012140627 A1 WO 2012140627A1 IB 2012051868 W IB2012051868 W IB 2012051868W WO 2012140627 A1 WO2012140627 A1 WO 2012140627A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the subject invention further provides isolated polypeptides comprising a sequence of amino acid residues corresponding to discrete portions of VSIGIO proteins, corresponding to the new junction and edge portions of VSIGIO variant (SEQ ID NO: 5).
- SEQ ID NO: 5 The unique sequence of the new junction of VSIGIO variant (SEQ ID NO: 5) is demonstrated in protein sequence alignment in Figure 2A.
- an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33-37, 40-46, 132, 155, 182-198, or variant thereof that possesses at least 95% sequence identity therewith, or a degenerative variant thereof.
- a method of immunotherapy in a patient comprising: in vivo or ex vivo tolerance induction, comprising administering effective amount of any of an isolated polypeptide as described herein, or a fusion protein as described herein; a nucleotide sequence as described herein; an expression vector as described herein; a host cell as described herein, or an antibody as described herein or a pharmaceutical composition, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells;
- the invention provides methods for tolerance induction, comprising in vivo or ex vivo treatment administration of effective amount of any one of isolated soluble LY6G6F, VSIGIO, TMEM25, LSR polypeptide, or a polypeptide comprising the extracellular domain of LY6G6F, VSIGIO, TMEM25, LSR, or fragment thereof, or a fusion thereof to a heterologous sequence, and/or a polyclonal or monoclonal antibody or antigen binding fragments and conjugates containing same, and/or alternative scaffolds, specific to any one of LY6G6F, VSIGIO, TMEM25 and/or LSR proteins, to a patient or to leukocytes isolated from the patient, in order to induce differentiation of tolerogenic regulatory cells, followed by ex- vivo enrichment and expansion of said cells and reinfusion of the tolerogenic regulatory cells to said patient.
- Figure 35 shows the dose dependency and mode of action of the effect of TMEM25-Ig (SEQ ID NO:25) in the R-EAE model in SJL mice.
- treatments were given from onset of disease remission (day 19) at 100, 30 or 10 microg/mouse i.p.
- the LY6G6F extracellular domain polypeptide consists essentially of the amino acid sequence of the IgV domain as set forth in any one of SEQ ID NOs: 81 and 96.
- the LY6G6F polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence.
- the signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence.
- the signal sequence of LY6G6F can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide.
- the signal peptide sequence that is used to replace the LY6G6F signal peptide sequence can be any known in the art.
- a soluble fragment of VSIG10 which may optionally be described as a first fusion partner in the below section on fusion proteins.
- Useful fragments are those that retain the ability to bind to their natural receptor or receptors and/or retain the ability to inhibit T cell activation.
- a VSIG10 polypeptide that is a fragment of full-length VSIG10 typically has at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the ability to bind its natural receptor(s) and/or of the ability to inhibit T cell activation as compared to full-length VSIG10.
- the VSIGIO polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence.
- the signal sequence is generally cleaved from the immature polypeptide to produce the mature polypeptide lacking the signal sequence.
- the signal sequence of VSIGIO can be replaced by the signal sequence of another polypeptide using standard molecule biology techniques to affect the expression levels, secretion, solubility, or other property of the polypeptide.
- the signal peptide sequence that is used to replace the VSIGIO signal peptide sequence can be any known in the art.
- the fragment is of at least about 160 up to 180 amino acids of the LSR protein extracellular domain, optionally including any integral value between 160 and 180 amino acids in length. More preferably, the fragment is about 108 or 145 or 170 amino acids.
- the LSR fragment protein according to at least some embodiments of the invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the LSR transmembrane domain.
- the LSR proteins contain an immunoglobulin domain within the extracellular domain, the IgV domain (or V domain), which is related to the variable domain of antibodies.
- Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
- amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxy lysine, isodesmosine, nor- valine, nor-leucine and ornithine.
- amino acid includes both D- and L-amino acids.
- the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
- a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
- Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
- regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
- the expression vectors according to at least some embodiments of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
- enzymes, and their cognate recognition sequences include Factor Xa, thrombin, PreScission, TEV and enterokinase.
- Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
- the expression vector encoding for the protein of the invention is a yeast expression vector.
- yeast expression vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
- the invention further provides a recombinant expression vector comprising a DNA molecule according to at least some embodiments of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to mRNA encoding for protein according to at least some embodiments of the invention.
- nucleic acid sequences encoding soluble LY6G6F, VSIGIO, TMEM25 and/or LSR proteins can be used in gene therapy for treatment of infectious disorders, and/or immune related disorders, and or cancer.
- high affinity for an IgG antibody refers to an antibody having a KD of 10-8 M or less, more preferably 10 -9 M or less and even more preferably 10 -10 M or less for a target antigen.
- high affinity binding can vary for other antibody isotypes.
- high affinity binding for an IgM isotype refers to an antibody having a KD of 10 -7 M or less, more preferably 10 -8 M or less.
- a human antibody comprises heavy or light chain variable regions that is "the product of” or “derived from” a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes.
- Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest.
- Antibodies that Bind to the Same Epitope as anti-LY6G6F, anti-VSIGlO, anti- TMEM25 or anti-LSR according to at least some embodiments of the invention.
- the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
- the Ms704, Ms705, and Ms709 FUT8.7- cell lines are created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22).
- mice with sufficient titers of anti- LY6G6F, anti-VSIGlO, anti-TMEM25 and/or anti-LSR human immunoglobulin can be used for fusions.
- Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each immunization may need to be performed. Between 6 and 24 mice are typically immunized for each antigen.
- HCo7 and HCol2 strains are used.
- both HCo7 and HCol2 transgene can be bred together into a single mouse having two different human heavy chain transgenes (HCo7/HCo 12).
- the KM Mouse. RTM. strain can be used.
- the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
- the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
- the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segments within the vector and the VK segment is operatively linked to the CL segment within the vector.
- Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site.
- TrxA thioredoxin
- Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
- gamma gamma.
- biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
- IL-1 interleukin-1
- IL-2 interleukin-2
- IL-6 interleukin-6
- GM-CSF granulocyte macrophage colony stimulating factor
- G-CSF granulocyte colony stimulating factor
- Fc alpha R or Fc epsilon R expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing LY6G6F, VSIGIO, TMEM25 and/or LSR, respectively.
- effector cells e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)
- PMNs polymorphonuclear cells
- the second polypeptide contains one or more domains of an immunoglobulin heavy chain constant region, preferably having an amino acid sequence corresponding to the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl, Cy2, Cy3 or Cy4 chain or to the hinge, CH2 and CH3 regions of a murine immunoglobulin Cy2a chain.
- SEQ ID NO: 70 provides exemplary sequence for the hinge, CH2 and CH3 regions of a human immunoglobulin Cyl.
- the fusion protein is a dimeric fusion protein.
- the conjugated molecule is another immunomodulatory agent that can enhance or augment the effects of the LY6G6F, VSIG10, TMEM25 and/or LSR fusion protein.
- the conjugated molecule is Polyethylene Glycol (PEG). Peptide or polypeptide linker domain
- Exemplary flexible peptides/polypeptides include, but are not limited to, the amino acid sequences Gly-Ser (SEQ ID NO: 159), Gly-Ser-Gly-Ser (SEQ ID NO:160), Ala-Ser (SEQ ID NO: 161), Gly-Gly- Gly-Ser (SEQ ID NO: 162), Gly4-Ser (SEQ ID NO: 163), (Gly4-Ser)2 (SEQ ID NO: 164), (Gly4-Ser)3 (SEQ ID NO: 165) and (Gly4-Ser)4 (SEQ ID NO: 166). Additional flexible peptide/polypeptide sequences are well known in the art.
- treating refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the above-described diseases, disorders or conditions. It also includes managing the disease as described above. By “manage” it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes and the like.
- Non-limiting examples of such known therapeutic agents or methods for treating IBD include immunosuppression to control the symptom, such as prednisone, Mesalazine (including Asacol, Pentasa, Lialda, Aspiro),azathioprine (Imuran), methotrexate, or 6-mercaptopurine, steroids, Ondansetron, TNF-a blockers (including infliximab, adalimumab golimumab, certolizumab pegol), Orencia (abatacept), ustekinumab (Stelara®), Briakinumab (ABT-874), Certolizumab pegol (Cimzia®), ITF2357 (givinostat), Natalizumab (Tysabri), Firategrast (SB-683699), Remicade (infliximab), vedolizumab (MLN0002), other drugs including GSK1605786 CCX282-B (Traficet
- Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
- “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
- the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
- dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
- An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
- the patient can be administered a prophylactic regime.
- Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
- the ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.
- this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
- a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
- One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
- kits based upon such diagnostic methods or assays. Also within the scope of the present invention are kits comprising the LY6G6F, VSIG10, TMEM25 and/or LSR protein or LY6G6F, VSIG10, TMEM25 and/or LSR conjugates or antibody compositions of the invention (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use.
- kits comprising the LY6G6F, VSIG10, TMEM25 and/or LSR protein or LY6G6F, VSIG10, TMEM25 and/or LSR conjugates or antibody compositions of the invention (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use.
- a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays.
- Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay ( IA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
- EIA enzyme immune assay
- ELISA enzyme-linked immunosorbent assay
- IA radioimmune assay
- Western blot assay e.g., Western blot assay
- slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
- a sample obtained from a subject can be contacted with the antibody
- the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
- solid supports include but are not limited to glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead.
- Antibodies can also be attached to a solid support.
- Radio-immunoassay In one version, this method involves precipitation of the desired substrate and in the methods detailed herein below, with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with 1125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
- a specific antibody and radiolabeled antibody binding protein e.g., protein A labeled with 1125
- a labeled substrate and an unlabelled antibody binding protein are employed.
- a sample containing an unknown amount of substrate is added in varying amounts.
- the decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
- the MED discovery engine was used to assess the expression of VSIG10 transcripts.
- Expression data for Affymetrix probe sets 220137_at representing the VSIG10 gene data is shown in Figure 3 (for all figures related to the MED discovery engine, a division was made into "A", B", etc for reasons of space only, so as to be able to show all probe results).
- the expression of VSIG10 transcripts detectable with the above probe sets was observed in several groups of cells from the immune system, mainly in leukocytes. In various cancer conditions, differential expression was observed, for example on CD10+ leukocytes from ALL (Acute Lymphoblastic Leukemia) and BM-CD34+cells from AML (Acute Myeloid Leukemia) cells.
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| JP2014504444A JP2014517685A (ja) | 2011-04-15 | 2012-04-16 | 免疫関連疾患およびがんの治療のためのポリペプチドおよびポリヌクレオチドならびにそれらの使用 |
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| MX2013011978A MX2013011978A (es) | 2011-04-15 | 2012-04-16 | Polipeptidos y polinucleotidos, y usos de los mismos para el tratamiento de transtornos inmunorelacionados y cancer. |
| BR112013026199A BR112013026199A2 (pt) | 2011-04-15 | 2012-04-16 | polipeptídeo isolado, proteína de fusão, sequência de ácidos nucleicos, vetor de expressão ou um vírus, célula recombinante, método de produção de um polipeptídeo com ectodomínio solúvel lsr, ou seu fragmento ou proteína de fusão, composição farmacêutica, uso de um anticorpo monoclonal ou policlonal ou um seu fragmento de ligação ao antígeno, uso do anticorpo ou do fragmento de ligação ao antígeno, uso de qualquer um de um polipeptídeo isolado, método de regulação por cima de citocinas, indução da expansão de células t, promoção da imunidade de células t específica antigênica e promoção da ativação das células t cd4+ e/ou cd8+ em um sujeito, método para potenciação de uma resposta imune secundária a um antígeno em um paciente, método de uso de pelo menos um de: um polipeptídeo isolado, método para tratamento ou prevenção de uma condição relacionada com o sistema imune, método para tratamento ou prevenção de uma doença infecciosa, método para diagnóstico de uma doença em um sujeito, método de produção de um polipeptídeo com ectodomínio solúvel tmem25, vsig10, ly6g6f, ou seu fragmento ou proteína de fusão, anticorpo monoclonal ou policlonal ou um seu fragmento de ligação ao antígeno, método de imunoterapia em um paciente e método para combinação de vacinação terapêutica com um antígeno em conjunto com a administração de qualquer um de um polipeptídeo |
| RU2013147915A RU2623161C2 (ru) | 2011-04-15 | 2012-04-16 | Полипептиды и полинуклеотиды и их применение для лечения иммунологических нарушений и рака |
| CN201280028877.0A CN103596974B (zh) | 2011-04-15 | 2012-04-16 | 多肽和多核苷酸及其用于治疗免疫相关失调和癌症的用途 |
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| RU2016147206A (ru) | 2018-10-19 |
| US20140044641A1 (en) | 2014-02-13 |
| IL249810A0 (en) | 2017-02-28 |
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| BR112013026199A2 (pt) | 2017-11-07 |
| SG194099A1 (en) | 2013-11-29 |
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| CN105601741A (zh) | 2016-05-25 |
| US20160347816A1 (en) | 2016-12-01 |
| NZ616344A (en) | 2015-11-27 |
| RU2623161C2 (ru) | 2017-06-22 |
| RU2013147915A (ru) | 2015-07-20 |
| AU2016222519A1 (en) | 2016-10-06 |
| EP2697256A1 (en) | 2014-02-19 |
| CN103596974B (zh) | 2016-08-31 |
| MX2013011978A (es) | 2015-06-10 |
| CA2830923A1 (en) | 2012-10-18 |
| JP2014517685A (ja) | 2014-07-24 |
| US20180194829A1 (en) | 2018-07-12 |
| US20220048972A1 (en) | 2022-02-17 |
| ZA201509074B (en) | 2017-11-29 |
| US9409987B2 (en) | 2016-08-09 |
| AU2016222519B2 (en) | 2017-09-28 |
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