CN107407673B - Novel glycan conjugate and its application method - Google Patents

Abstract

The present invention relates to vaccine, antibody and/or immunogenic conjugates composition that targeting SSEA3/SSEA4/GloboH related antigen determines base (naturally and through modifying), induces antibody and/or binding fragment is generated for adjusting the synthesis of ball series glycosyl sphingolipid.The present invention relates to adjustable ball series glycosyl sphingolipid synthetic method and compositions.In specific words, the present invention relates to carbohydrase inhibitors compound synthesized of the ball series glycosyl sphingolipid SSEA3/SSEA4/GloboH in adjustable Biosynthetic pathway and combinations thereof and application methods；In specific words, α-Isosorbide-5-Nitrae-galactosyltransferase in carbohydrase inhibitors targeting ball series synthesis path；β -1,4-N- acetyl galactose based transferase-I；Or β -1,3- galactosyltransferase-V.In addition, the present invention also relates to the method for being treated or being detected hyperproliferative disease and/or symptom using composition as described herein.

Description

Novel glycan conjugate and its application method

Related application

The present invention advocates the priority of United States provisional application serial number 62/107,378 according to 35 U.S.C.119 (e), Apply on January 24th, 2015, entitled novelty glycan conjugate and its application method.Its content is with the side of being cited in full text Formula is incorporated herein.

Technical field

The present invention relates to adjustable ball series glycosyl sphingolipid synthetic method and compositions.In specific words, the present invention relates to can Adjust carbohydrase inhibitors compound of ball series glycosyl sphingolipid synthesis in Biosynthetic pathway and combinations thereof and application method；It is special Surely it is sayed, carbohydrase inhibitors target α-Isosorbide-5-Nitrae-galactosyltransferase in ball series synthesis path；β -1,4-N- acetyl gala Glycosyl transferase-I；Or β -1,3- galactosyltransferase-V.In addition, the present invention also relates to targeting SSEA3/SSEA4/GloboH Related antigen determines vaccine, antibody and/or the immunogenic conjugates composition of base (naturally and through modifying), induces antibody And/or binding fragment is generated for adjusting the synthesis of ball series glycosyl sphingolipid.In addition, the present invention is also related to using combination as described herein The method of object treatment or detecting hyperproliferative disease and/or symptom.

Background technique

Carbohydrate Antigens GloboH, phase specificity embryonal antigen -3 (SSEA3) and phase specificity embryonal antigen -4 (SSEA4) closely related to one another in structure or function.GloboH, SSEA3 and SSEA4 are ball series glycosyl sphingolipid, wherein SSEA3 For the non-fucosylated pentasaccharides predecessor structure of GloboH, SSEA4 is connected to the non-of SSEA3 galactolipin for sialic acid α 2-3 and goes back The sialylated SSEA3 at former end.

Identify phase specificity embryonal antigen -3 (SSEA3) first and according to the rat being immunized through 4 to 8 cell stage Development of Mouse Embryos In the reactivity of generated IgM monoclonal antibody define.Embryo is (from egg mother cell before the implantation of this monoclonal antibody and all mouse Until the early blastocyst phase) react, wherein the expression in its primitive endoderm after the implants is more limited.SSEA3 Antigenic determinants are determined to the carbohydrate being present on glycolipid and glycoprotein；It is red in mankind's teratocarcinoma cells and the mankind Also SSEA3 antigenic determinants are found on blood cell.In the structure that one group separates from 2102Ep mankind's teratocarcinoma cells strain, SSEA3 antibody has highest affinity for Gal β (1-3) GalNAc β (1-3) Gal α (1-4) Gal β (1-4) Glc β (1) Cer. This structure is also known as Gb5 (five glycosyl ceramide of galactosyl-red blood cell glucosides rouge or globuli cell).

When β -1,3- galactoside based transferase V (β 3-GalT-V) makes galactolipin be transferred to the N- second of red blood cell glucosides rouge When forming Gb5 or galactosyl-red blood cell glucosides rouge on acyl galactosamine, the synthesis of SSEA3 occurs.Have determined that Hematopoietic Stem is thin Born of the same parents or interstital stem cell do not express SSEA3.Immortalization lymph node lymphocyte based on primary patients with lung cancer, generate fusion tumor and Select antibody-secreting pure lines；Then the two (J309 and D579) thus in equal pure lines generates identification SSEA3 antigenic determinants Monoclonal antibody.Antibody identifies on several tumor cell lines (including lung cancer and breast cancer cell strain and teratocarcinoma cells strain) SSEA3；In immune adherence analysis, identical as J309 and D579 antibody, rodent single plant SSEA3 antibody is (also known as MC631 it) reacts for these cell strains.Also in Testicular Germ Cell Tumors and breast cancer and BCSC (breast cancer stem cell) Upper discovery SSEA3.

Chang et al. inspects expression of the SSEA3 in normal tissue using the micro- array of tissue, and reason is it in cancer Outer position and substantially unknown (A.2008 19 Aug Proc Natl Acad Sci U S of development；105(33):11667- 72,Expression of GloboH and SSEA3 in breast cancer stem cells and the involvement of fucosyl transferases 1 and 2 in GloboH synthesis).Study group's hair Existing SSEA3 is expressed in the normal epithelial of colon, esophagus, small intestine, kidney, prostate, rectum, skin, testis, thymus gland and cervix On.Expression is only located on the top surface of epithelial cell or is located in cytoplasm, these positions are considered as that immune system is restricted Or non-accessible site.It is anti-only for GloboH in the experiment for using univalent vaccine of the KLH in conjunction with GloboH to mouse Primary raw antibody response.When α-GalCer is added as adjuvant, general antibody yield increases and mouse generates and is directed to The multi-strain antibody of GloboH, SSEA3 and SSEA4 antigenic structure cannot generate vaccine inoculation in the case where lacking adjuvant.This knot Fruit shows that SSEA3, GloboH and SSEA4 can be expected to the target as cancer vaccine and can become target simultaneously.

However, most tumour related carbohydrate antigen has undesirable immunogenicity and has developed a variety of Method enhances the immune response of the vaccine based on carbohydrate, including in conjunction with carrier protein matter, joint utilize non-natural The bonded immunologic adjuvant administration of glucosides, antigen of gathering together, unimolecule polyvaccine or heteroglycan polyvaccine.It is tactful using these, Several vaccines based on carbohydrate for the significant immune response of target glycan structures can be induced for cancer by designing Therapy and enter clinical test.Wherein, between sick time and between overall survival rate, Theratope and GMK combine adjuvant The clinical test of QS-21, which fails to generate, counts significant sex differernce.Both vaccines are likely to that stable T will not be induced in patients Cell dependent immune reaction.Specifically, Theratope and GMK induce the IgM of high-content in patients, but not induce Stronger IgG immune response, this is the main problem in the vaccine development based on carbohydrate.

Previous research are shown, can effectively be induced the modification of Carbohydrate Antigens structure (MCAS) high-caliber immune Reaction.For example, in the modification research of the capsular polysaccharide of B group meningitis cocci, α-(2,8) connecting-type poly sialic acid (PSA) the high antibody response that N- acetyl group is replaced through N- propiono and this modification induces not only identifies the poly- saliva of N- propiono Acid, and identify natural N- acetyl group poly sialic acid.It is right in order to generate the high antibody titer for being directed to modified forms and native form STn and GM3 antigen application similar approach.The result shows that N- phenethyl, N- acetyl fluoride base or bis- acetyl fluoride of N- on glycan antigen Base modification can improve immunogenicity.In addition, schulz (Schultz) study group reports, p- nitro Phenylalanine is incorporated to tumour Immunological tolerance can be destroyed in necrosis factor-alpha (TNF-α) and induces more antibody responses for TNF-α.Use glycan as Although antigen has reached some progress, most of situation is the nitrogen base of disaccharides (STn), trisaccharide (GM3) and poly sialic acid (PSA) Modification；And some cases are based on fluorination MUC1 glycopeptide antigen.

Summary of the invention

The present invention is based on the discovery that the phase specificity embryonal antigen (SSEA3 with certain groups disclosed herein And SSEA4) modification induce can respectively specifically identify SSEA3 and SSEA4 stabilization IgG antibody reaction.Comprising such non- The antibody that the immunogenic composition of natural glycan moiety is induced can mediate the complement dependent cellular for tumour cell Cytotoxicity.

Therefore, the present invention shows the design of the antibody for SSEA3 and/or SSEA4 for treating cancer.The present invention is also Show the compounds being made of modified Carbohydrate Antigens (SSEA3 and SSEA4), gathering comprising such compound Glycoconjugate and its immunogenic composition and vaccine.

The present invention also provides using synthesis glycan conjugates for therapy as described herein or mitigates hyperproliferative disease (such as Cancer) method.

In addition, the present invention, which also relates to targeting SSEA3/SSEA4/GloboH related antigen, determines base (naturally and through modifying) Vaccine and/or immunogenic conjugates composition, can induce antibody and/or binding fragment is generated for adjusting ball series sheath sugar Rouge synthesis.In addition, the present invention also relates to be treated or detected hyperproliferative disease and/or symptom using composition as described herein Method.

Therefore, the present invention shows the design of the antibody for SSEA3 for treating cancer.The present invention is also shown by through repairing Compounds, the glycan conjugate comprising such compound of Carbohydrate Antigens (the SSEA3, SSEA4) composition of decorations, and Its immunogenic composition and vaccine.

In an aspect, the present invention provides formula (I) compound:

Or its salt, wherein X₁、R¹、R²、R³、R⁴、R⁵、R⁶And L is as described herein.In certain embodiments, formula (I) compound Suitable for preparing the immunogenic composition for treating cancer.

In another aspect, the present invention provides formula (II) compound:

Or its salt, wherein X₁、R¹、R²、R³、R⁸、R⁹、R¹⁰、R¹¹And R_NAs described herein.In certain embodiments, formula (II) Compound is suitable for preparing the immunogenic composition for treating cancer.

In another aspect, the present invention provides a kind of immunogenic composition, and comprising (a) include carrier and one or more are poly- The glycan conjugate of sugar, and optionally existing (b) adjuvant,

Wherein: each of one or more glycan via connector in conjunction with carrier, with formula (III) or (IV):

Wherein X₁、R¹、R²、R³、R⁴、R⁵、R⁶、R⁸、R⁹、R¹⁰、R¹¹, L and R_NAs described herein.

In certain aspects, it is contemplated that containing there are three types of any one of glycan (SSEA3, SSEA4 and GloboH) and the like Or any vaccine construct of the combination of any ratio of more persons can be connected to carrier.

Wherein n can be integer 1 to 10；

Wherein glycan can be selected from the group being made of Formulas I, II, III and IV；

If wherein n is 2 or greater than 2, each glycan can be with another glycan or aspartyl peptide on aspartyl peptide On different glycan it is identical.

In some embodiments, glycan can be selected from the group being made of SSEA3, SSEA4 and GloboH.

In some embodiments, illustrative multivalence construct can are as follows:

The wherein R on each glycan moiety¹、R²、R³、R⁴、R⁵、R⁶And L can be identical or different.

In certain embodiments, immunogenic composition of the invention includes adjuvant.It is suitable for the invention illustrative assistant Agent is as described herein.

In certain embodiments, immunogenic composition can induce the immune response for cancer cell in individual.? In some embodiments, cancer cell is selected from the group that is made up of: brain cancer cell, lung carcinoma cell, breast cancer cell, cancer cell of oral cavity, Esophageal cancer cell, stomach cancer cell, liver cancer cells, cholangiocarcinoma cell, pancreatic cancer cells, colon cancer cell, renal carcinoma cells, osteocarcinoma Cell, skin cancer cell, cervical cancer cell, ovarian cancer cell and prostate gland cancer cell.

In certain embodiments, immune response include be specifically bound to one or more be selected from by GloboH, SSEA3 and The generation of the antibody of the antigen of the group of SSEA4 composition.In certain embodiments, target cancer cell or cancer stem cell table are developed One or more of expressed GloboH, SSEA3 and SSEA4 and initiation complement-dependent cytotoxicity and/or antibody on face Dependent cells mediating cytotoxicity kills this isocellular antibody.In certain embodiments, antibody mainly includes IgG antibody.? In some embodiments, immunogenic composition provided in this article mainly induces IgG1, IgG2b, IgG2c and IgG3.

In addition, the present invention is shown for monoclonal antibody and bonding pad caused by immunogenic composition as described herein Section.

In one embodiment, antibody is human antibodies.

In one embodiment, antibody is humanization antibody.

In one embodiment, one or more of antibody specificity targeting SSEA4, SSEA3 or GloboH.

In one embodiment, antibody specificity targets SSEA3.

In one embodiment, antibody specificity targets SSEA4.

In one embodiment, antibody is the homogeneous antibody with double feeler glycan, and double feeler glycan are through two salivas Liquid acid blocks in such a way that α -2,6- is bonded.

In an aspect, the present invention provides a kind of medical composition, it includes a effective amount of selectively targeted SSEA4, The antibody or antigen-binding fragment and pharmaceutically acceptable carrier of one or more of SSEA3 or GloboH.

In one embodiment, medical composition includes to target SSEA4, SSEA3 and/or GloboH glycan each independently One or more of antibody and/or its binding fragment combination.

In one embodiment, medical composition is suitable for treating cancer, infectious diseases and/or inflammatory diseases.

In one embodiment, medical composition includes the antibody or its binding fragment with general double feeler N- glycan, General double feeler N- glycan block in such a way that α -2,6- is bonded through sialic acid.

In another aspect, the present invention is provided comprising immunogenic composition as described herein and pharmaceutically acceptable The cancer vaccine of excipient.

In another aspect, the method that the present invention provides the cancer for the treatment of individual and/or reduces the risk of cancer of individual, packet Containing the immunogenic composition or cancer vaccine as described herein to the individual administration therapeutically effective amount for needing it.

Treatment can reduce tumor size, eliminate malignant cell, prevention metastasis of cancer, prevention recurrence, reduce or kill dissemination Cancer extends survival period and/or extends the time of tumor and cancer progress.

In some embodiments, treatment is further contained in administration immunogenic composition as described herein or cancer vaccine Before, during or after to the individual administration another kind therapy.In some embodiments, another therapy is to use chemotherapy Agent treatment.In some embodiments, another therapy is radiation-therapy.

Another aspect of the invention shows a kind of method with the vaccine inoculation mammal for cancer, includes to lactation Animal administration pharmacologically a effective amount of immunogenic composition as described herein or cancer vaccine.

In some embodiments, mammal is the mankind.In some embodiments, subcutaneous administration immunogene as described herein Property composition or cancer vaccine.

The example of cancer includes but is not limited to the cancer of the brain, lung cancer, breast cancer, carcinoma of mouth, cancer of the esophagus, gastric cancer, liver cancer, bile duct Cancer, cancer of pancreas, colon cancer, renal cancer, cervix cancer, oophoroma and prostate cancer.In some embodiments, cancer be the cancer of the brain, Lung cancer, breast cancer, oophoroma, prostate cancer, colon cancer or cancer of pancreas.

In another aspect, the present invention provides the method for synthesizing the compounds of this invention as described herein.

In another aspect, the present invention shows the side for preparing immunogenic composition or cancer vaccine as described herein Method.

The details of certain embodiments of the present invention illustrates in this article.Other features, target and advantage of the invention according to Embodiment, schema, example and claim will be evident.

Detailed description of the invention

Fig. 1: the Biosynthetic pathway of ball series glycosyl sphingolipid.

Fig. 2: from the mouse of the SSEA4-CRM197 or SSEA4-Gc-CRM197 immunity inoculation of different epitope ratios In it is collected through inducing GloboH-IgG antibody.

Fig. 3 A: primary SSEA4 and all eight kinds of SSEA4 analogs can induce and be directed to when being applied in combination with Gal-C34 The IgG antibody of SSEA4.

Fig. 3 B: primary SSEA4 and all eight kinds of SSEA4 analogs can induce and be directed to when being applied in combination with Gal-C34 The IgM antibody of SSEA4.

Fig. 4 A: primary SSEA4 and all eight kinds of SSEA4 analogs can induce and be directed to when being applied in combination with Glc-C34 The IgG antibody of SSEA4.

Fig. 4 B: primary SSEA4 and all eight kinds of SSEA4 analogs can induce and be directed to when being applied in combination with Glc-C34 The IgM antibody of SSEA4.

Fig. 5: glycan-protein combination method influences immune response.

Specific embodiment

The present invention is based on following surprising discovery: phase specificity embryonal antigen (SSEA3 and SSEA4) is through certain base group modifications The stabilization IgG antibody reaction of specific recognition SSEA3 and SSEA4 respectively can be induced.

In some instances, the modification of SSEA3 includes the fluorine on one or more positions of the glucose of SSEA3, repeatedly nitrogen base Or O- phenyl.In some instances, the fluorine of SSEA3 modified on one or more positions comprising non-reducing end galactolipin, repeatedly nitrogen Base or O- phenyl.In some instances, the fluorine on one or more positions for modifying the glucose comprising SSEA4 of SSEA4, repeatedly nitrogen Base or O- phenyl.In some instances, the modification of SSEA4 includes the fluorine on one or more positions of sialic acid residues, repeatedly nitrogen base Or O- phenyl.

Reducing end and/or non-reducing end, which is described herein, has the SSEA3 analog and SSEA4 analog of modification.Compared to Primary SSEA3 and SSEA4, such SSEA3 and SSEA4 analog can induce stronger immune response, and (such as induction is for SSEA3 And/or the IgG antibody of SSEA4).The antibody that immunogenic composition comprising such non-natural glycan moiety is induced can be situated between Cytotoxicity of the guide pin to the complement dependent cellular of tumour cell.

Chemistry definition

The definition of specific functional group and the technical terms of chemistry is more fully hereinafter described.Chemical element is according to Handbook of The periodic table of elements (CAS editions) of cover identifies in Chemistry and Physics the 75th edition, and specific functional group is generally It defines as described therein.In addition, the General Principle of organic chemistry and particular functional part and reactivity are described in Thomas Sorrell,Organic Chemistry,University Science Books,Sausalito,1999； Smith and March, March's Advanced Organic Chemistry, the 5th edition, John Wiley&Sons, Inc., New York,2001；Larock,Comprehensive Organic Transformations,VCH Publishers, Inc.,New York,1989；And Carruthers, Some Modern Methods of Organic Synthesis, the 3rd Version, Cambridge University Press, Cambridge, 1987.In addition, illustrative glycan and antibody method are described in In US20100136042, US20090317837 and US20140051127 of Wong et al., the disclosure of each of which is to draw Mode is incorporated herein.

Compound as described herein can include one or more of asymmetric center, and therefore can be with various isomeric forms (examples Such as enantiomter and/or diastereoisomer) exist.For example, compound as described herein can be in individual enantiomerisms Body, diastereoisomer or geometric isomer form, or can spatially isomers form of mixtures, including racemic mixture And the wherein mixture of one or more stereoisomer enrichments.Isomers can utilize those who familiarize themselves with the technology known method (packet Include the formation and crystallization to palm property high pressure liquid chromatography (HPLC) (HPLC) and to palm property salt) and separated from mixture；Or it can be by not Symmetrical synthesis is to prepare preferred isomers.See, for example, Jacques et al., Enantiomers, Racemates and Resolutions(Wiley Interscience,New York,1981)；Wilen et al., Tetrahedron 33:2725 (1977)；Eliel,Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；And Wilen, Tables of Resolving Agents and Optical Resolutions page 268 (E.L.Eliel is compiled, Univ.of Notre Dame Press,Notre Dame,IN 1972).In addition the present invention covers different in other are substantially free of The individual isomeric forms of structure body and alternatively in various isomers form of mixtures compound described herein.

When enumerator range, it is desirable to cover each value and the subrange within the scope of this.For example, " C1-6 " is intended to contain Cover C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5 and C5-6.

" alkyl " refers to straight chain or branched chain saturated hydrocarbyl (" C1-20 alkyl ") with 1 to 20 carbon atom.Some In embodiment, alkyl has 1 to 10 carbon atom (" C1-10 alkyl ").In some embodiments, alkyl has 1 to 9 carbon original Sub (" C1-9 alkyl ").In some embodiments, alkyl has 1 to 8 carbon atom (" C1-8 alkyl ").In some embodiments In, alkyl has 1 to 7 carbon atom (" C1-7 alkyl ").In some embodiments, alkyl has 1 to 6 carbon atom (" C1-6 Alkyl ").In some embodiments, alkyl has 1 to 5 carbon atom (" C1-5 alkyl ").In some embodiments, alkyl has There is 1 to 4 carbon atom (" C1-4 alkyl ").In some embodiments, alkyl has 1 to 3 carbon atom (" C1-3 alkyl ").? In some embodiments, alkyl has 1 to 2 carbon atom (" C1-2 alkyl ").In some embodiments, alkyl has 1 carbon original Sub (" C1 alkyl ").In some embodiments, alkyl has 2 to 6 carbon atoms (" C2-6 alkyl ").The example packet of C1-6 alkyl Include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), normal-butyl (C4), third butyl (C4), the second butyl (C4), isobutyl group (C4), n-pentyl (C5), 3- amyl (C5), amyl (C5), neopentyl (C5), 3- methyl -2- butyl (C5), Third amyl (C5) and n-hexyl (C6).Other examples of alkyl include n-heptyl (C7), n-octyl (C8) and its similar group. Unless otherwise stated, otherwise each example of alkyl is independently optionally substituted, that is, is unsubstituted the (" alkane being unsubstituted Base ") or through one or more substituent groups substitution (" alkyl being substituted ").In certain embodiments, alkyl is the C1- being unsubstituted 10 alkyl (such as-CH₃).In certain embodiments, alkyl is the C1-10 alkyl being substituted.

" alkenyl " refer to 2 to 20 carbon atoms, one or more carbon-to-carbon double bonds and without ginseng key straight chain or branch's chain hydrocarbon Base (" C2-20 alkenyl ").In some embodiments, alkenyl has 2 to 10 carbon atoms (" C2-10 alkenyl ").In some implementations In example, alkenyl has 2 to 9 carbon atoms (" C2-9 alkenyl ").In some embodiments, alkenyl have 2 to 8 carbon atoms (" C2-8 alkenyl ").In some embodiments, alkenyl has 2 to 7 carbon atoms (" C2-7 alkenyl ").In some embodiments, alkene Base has 2 to 6 carbon atoms (" C2-6 alkenyl ").In some embodiments, alkenyl has 2 to 5 carbon atom (" C2-5 alkene Base ").In some embodiments, alkenyl has 2 to 4 carbon atoms (" C2-4 alkenyl ").In some embodiments, alkenyl has 2 To 3 carbon atoms (" C2-3 alkenyl ").In some embodiments, alkenyl has 2 carbon atoms (" C2 alkenyl ").One or more Carbon-to-carbon double bond can internally positioned (such as 2- cyclobutenyl) or end (such as 1- cyclobutenyl).The example of C2-4 alkenyl includes ethylene Base (C2), 1- acrylic (C3), 2- acrylic (C3), 1- cyclobutenyl (C4), 2- cyclobutenyl (C4), butadienyl (C4) and its class Like group.The example of C2-6 alkenyl include aforementioned C2-4 alkenyl and pentenyl (C5), pentadienyl (C5), hexenyl (C6) and It is similar to group.Other examples of alkenyl include heptenyl (C7), octenyl (C8), sarohornene base (C8) and its similar group. Unless otherwise stated, otherwise each example of alkenyl is independently optionally substituted, also that is, being unsubstituted the (" alkene being unsubstituted Base ") or through one or more substituent groups substitution (" alkenyl being substituted ").In certain embodiments, alkenyl is the C2- being unsubstituted 10 alkenyls.In certain embodiments, alkenyl is the C2-10 alkenyl being substituted.

" alkynyl " refers to 2 to 20 carbon atoms, one or more carbon-to-carbon triple bonds and optionally existing one or more are double The straight chain or branch's chain alkylene (" C2-20 alkynyl ") of key.In some embodiments, alkynyl has 2 to 10 carbon atom (" C2-10 Alkynyl ").In some embodiments, alkynyl has 2 to 9 carbon atoms (" C2-9 alkynyl ").In some embodiments, alkynyl has There is 2 to 8 carbon atoms (" C2-8 alkynyl ").In some embodiments, alkynyl has 2 to 7 carbon atoms (" C2-7 alkynyl ").? In some embodiments, alkynyl has 2 to 6 carbon atoms (" C2-6 alkynyl ").In some embodiments, alkynyl has 2 to 5 carbon Atom (" C2-5 alkynyl ").In some embodiments, alkynyl has 2 to 4 carbon atoms (" C2-4 alkynyl ").In some embodiments In, alkynyl has 2 to 3 carbon atoms (" C2-3 alkynyl ").In some embodiments, alkynyl has 2 carbon atom (" C2 alkynes Base ").One or more carbon-to-carbon triple bonds can internally positioned (such as 2- butynyl) or end (such as 1- butynyl).C2-4 alkynyl Example includes but is not limited to acetenyl (C2), 1- propinyl (C3), 2-propynyl (C3), 1- butynyl (C4), 2- butynyl (C4) and its similar to group.The example of C2-6 alkenyl include aforementioned C2-4 alkynyl and pentynyl (C5), hexin base (C6) and its Similar group.Other examples of alkynyl include heptynyl (C7), octynyl (C8) and its similar group.Unless otherwise stated, no Then each example of alkynyl is independently optionally substituted, that is, is unsubstituted (" alkynyl being unsubstituted ") or is taken through one or more Replace (" alkynyl being substituted ") for base.In certain embodiments, alkynyl is the C2-10 alkynyl being unsubstituted.In certain implementations In example, alkynyl is the C2-10 alkynyl being substituted.

" heterocycle " or " heterocycle " refers to 3 yuan to the 10 yuan non-aromatic ring systems with ring carbon atom and 1 to 4 ring hetero atom Group, wherein each hetero atom is independently selected from nitrogen, oxygen, sulphur, boron, phosphorus and silicon (" 3 to 10 circle heterocyclic ring base ").In some embodiments In, hetero atom is independently selected from nitrogen, sulphur and oxygen.In the heterocycle containing one or more nitrogen-atoms, when valence mumber allows, tie point It can be carbon or nitrogen-atoms.Heterocycle can be monocycle (" monocyclic heterocycles base ") or condensed, bridging or spiral ring system, such as bicyclic system (" bicyclic heterocyclic radical "), and can be unsaturated for saturation or part.Heterocycle bicyclic system can include one in one or two ring Or multiple hetero atoms." heterocycle " also includes the ring system that wherein heterocycle as defined above and one or more carbocylic radicals condense System, wherein tie point is located on carbocylic radical or heterocycle；Or in which heterocycle as defined above and one or more aryl or heteroaryl The condensed loop system of base, wherein tie point is located on heterocycle, and in tiiese cases, and ring members number continues to indicate in heterocyclic ring system Ring members number.Unless otherwise stated, otherwise each example of heterocycle is independently optionally substituted, also that is, be unsubstituted (" The heterocycle being unsubstituted ") or through one or more substituent groups substitution (" heterocycle being substituted ").In certain embodiments, miscellaneous Ring group is 3 to 10 circle heterocyclic ring bases being unsubstituted.In certain embodiments, heterocycle is 3 to 10 circle heterocyclic ring bases being substituted.

" aryl " refers to that monocycle or polycyclic (such as bicyclic or tricyclic) 4n+2 aromatic ring systems (such as are shared in cyclic annular array 6,10 or 14 pi-electrons) group, there is in the aromatic ring systems 6 to 14 ring carbon atoms and zero heteroatoms (" C6-14 Aryl ").In some embodiments, aryl has 6 ring carbon atoms (" C6 aryl "；Such as phenyl).In some embodiments, Aryl has 10 ring carbon atoms (" C10 aryl "；Such as naphthalene, such as 1- naphthalene and 2- naphthalene).In some embodiments, fragrant Base has 14 ring carbon atoms (" C14 aryl "；Such as anthryl)." aryl " also include wherein aryl rings as defined above with The loop system that one or more carbocylic radicals or heterocycle condense, wherein linking group or tie point are located in aryl rings, and at these In the case of, carbon atom number continues to indicate the carbon atom number in aryl loop system.Unless otherwise stated, otherwise each example of aryl is only It is on the spot optionally substituted, that is, is unsubstituted (" aryl being unsubstituted ") or (" is substituted through the substitution of one or more substituent groups Aryl ").In certain embodiments, aryl is the C6-14 aryl being unsubstituted.In certain embodiments, aryl is to be substituted C6-14 aryl.

As herein defined as the alkyl of divalent bridging group, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl and miscellaneous In addition aryl is stretched base using suffix-and is referred to, for example, stretch alkyl, stretch alkenyl, stretch alkynyl, stretch carbocylic radical, stretch heterocycle, stretch aryl and Stretch heteroaryl.

Term " alkoxy " or " alkyl oxy " refer to-O- alkyl, and wherein alkyl optionally passes through alkane as herein defined Base replaces.The example of alkoxy includes but is not limited to methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, different Butoxy, the second butoxy and third butoxy.

Term " aryloxy group " refers to-O- aryl, and wherein aryl optionally replaces through aryl as herein defined.

As used herein, term " being optionally substituted " refers to the part for being substituted or being unsubstituted.

Alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl and heteroaryl as herein defined are optionally substituted (such as the alkyl of " being substituted " or " being unsubstituted ", the alkenyl of " being substituted " or " being unsubstituted ", " being substituted " or " without taking The alkynyl in generation ", the carbocylic radical of " being substituted " or " being unsubstituted ", the heterocycle of " being substituted " or " being unsubstituted ", " being substituted " " being unsubstituted " aryl or " being substituted " or " being unsubstituted " heteroaryl).In general, term " substitution ", whether It is no to be located at before term " optionally ", mean at least one hydrogen (such as carbon or nitrogen-atoms) being present on group through allowing Substituent group (such as replace when generate stable compound (such as cannot spontaneously undergo conversion (such as reset, be cyclized, elimination or its He react) compound) substituent group) displacement.Unless otherwise specified, the group of " being substituted " the group one or Multiple positions that may replace are with substituent group, and when more than one position in any given structure is substituted, and substituent group is each Position is identical or different.Term " being substituted " expection includes that all through organic compound allow substituent group, cause to stablize chemical combination Any substituent group described herein that object is formed replaces.The present invention covers any and all such combination to obtain stable chemical combination Object.For purposes of the present invention, hetero atom (such as nitrogen) can have hydrogen substituent group and/or meet hetero atom valence mumber and cause to stablize Any suitable substituent group as described herein that part is formed.

" halogen " or " halogen " refers to fluorine (fluorine-based ,-F), chlorine (chloro ,-Cl), bromine (bromo ,-Br) or iodine (iodo ,-I).

As used herein, " acyl group " refers to selected from the part of group being made up of :-C (=O) Raa ,-CHO ,- CO₂Raa ,-C (=O) N (Rbb)₂,-C (=NRbb) Raa ,-C (=NRbb) ORaa ,-C (=NRbb) N (Rbb)₂,-C (=O) NRbbSO₂Raa ,-C (=S) N (Rbb)₂,-C (=O) SRaa and-C (=S) SRaa, wherein Raa and Rbb are as defined herein.

When valence mumber allows, nitrogen-atoms can be substituted or be unsubstituted, and including level-one, second level, three-level and level Four nitrogen-atoms. Illustrative nitrogen-atoms substituent group includes but is not limited to hydrogen ,-OH ,-ORaa ,-N (Rcc)₂,-CN ,-C (=O) Raa ,-C (=O) N (Rcc)₂、-CO₂Raa、-SO₂Raa ,-C (=NRbb) Raa ,-C (=NRcc) ORaa ,-C (=NRcc) N (Rcc)₂、-SO₂N (Rcc)₂、-SO₂Rcc、-SO₂ORcc ,-SORaa ,-C (=S) N (Rcc)₂,-C (=O) SRcc ,-C (=S) SRcc ,-P (=O)₂Raa ,-P (=O) (Raa)₂,-P (=O)₂N(Rcc)₂,-P (=O) (NRcc)₂, C1-10 alkyl, C1-10 perhaloalkyl radical, C2-10 Alkenyl, C2-10 alkynyl, C3-10 carbocylic radical, 3-14 circle heterocyclic ring base, C6-14 aryl and 5-14 unit's heteroaryl, or it is connected to nitrogen original Two Rcc groups of son connect and form 3-14 circle heterocyclic ring base or 5-14 unit's heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbon Ring group, heterocycle, aryl and heteroaryl independently replace through 0,1,2,3,4 or 5 Rdd group and wherein Raa, Rbb, Rcc and Rdd is as hereinbefore defined.

In certain embodiments, the substituent group being present on oxygen atom is that oxygen protecting group (is also known as " hydroxyl guarantor herein Protect base ").Oxygen protecting group includes but is not limited to-Raa ,-N (Rbb)₂,-C (=O) SRaa ,-C (=O) Raa ,-CO₂Raa、-C (=O) N (Rbb)₂,-C (=NRbb) Raa ,-C (=NRbb) ORaa ,-C (=NRbb) N (Rbb)₂,-S (=O) Raa ,- SO₂Raa、-Si(Raa)₃、-P(Rcc)₂、-P(Rcc)₃,-P (=O)₂Raa ,-P (=O) (Raa)₂,-P (=O) (ORcc)₂、-P (=O)₂N(Rbb)₂And-P (=O) (NRbb)₂, wherein Raa, Rbb and Rcc are as defined herein.Oxygen protecting group is in technique In it is well known and including Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, 3rd edition, John Wiley&Sons, oxygen blocking group described in 1999, the document is incorporated herein by reference.

Illustrative oxygen blocking group includes but is not limited to methyl, methoxy (MOM), methylthiomethyl (MTM), Tributyl sulfidomethyl, (pheiiyldimetliyl silylation) methoxy (SMOM), benzyloxymethyl (BOM), to methoxybenzene Methoxy (PMBM), (4- methoxyphenoxy) methyl (p-AOM), o-methoxyphenol methyl (GUM), third butoxy Methyl, 4- amylene oxygroup methyl (POM), silanyloxymethyl, 2- methoxvethoxvmethvl (MEM), 2,2,2- tri-chloroethoxy Ylmethyl, bis- (2- chloroethoxy) methyl, 2- (trimethylsilyl) ethoxyl methyl (SEMOR), tetrahydro piperazine mutter base (THP), 3- bromine tetrahydro piperazine mutter base, 1- methoxycyclohexyl, 4- methoxyl group tetrahydro piperazine of the thio piperazine of base, tetrahydro of muttering is muttered base (MTHP), 4- methoxy Mutter base, the thio piperazine of 4- methoxyl group tetrahydro of the thio piperazine of base tetrahydro is muttered base S, S- dioxide, 1- [(the chloro- 4- methyl of 2-) phenyl] -4- Methoxy piperide -4- base (CTMP), 1,4- dioxanes -2- base, tetrahydrofuran base, tetrahydro thio-furan base, 2,3,3a, 4,5,6, 7,7a- octahydro -7,8,8- trimethyl -4,7- methanol benzofuran -2- base, 1- ethoxyethyl group, 1- (2- chloroethoxy) ethyl, 1- methyl-1-methoxy ethyl, 1- methyl-1-benzyloxy ethyl, 1- methyl-1-benzyloxy-2- fluoro ethyl, 2,2,2- tri- Chloroethyl, 2- trimethylsilyl ethyl, 2- (phenylselenyl) ethyl, third butyl, allyl, rubigan, to methoxy Base phenyl, dinitrophenyl group, benzyl (Bn), to mehtoxybenzyl, 3,4- dimethoxy benezene, ortho-nitrophenyl first Base, p-nitrophenyl methyl, to halogen benzyl, 2,6- dichlorobenzyl, to cyanobenzyl, to phenylphenylmethyl, 2- pyridine It is methyl, 4- picolyl, 3- methyl -2- picolyl N- oxonium ion base, benzhydryl, right, to '-dinitro benzhydryl, 5- Dibenzocycloheptyl, trityl, Alpha-Naphthyl diphenyl methyl, p-methoxyphenyl diphenyl methyl, two are (to methoxybenzene Base) phenyl methyl, three (p-methoxyphenyl) methyl, 4- (4'- Bromophenac rLl phenyl) benzhydryl, 4,4', 4 "- Ginseng (bis- chlorophthalimido phenyl of 4,5-) methyl, 4,4', 4 "-ginseng (jerusalem artichoke glycosyl phenyl) methyl, 4,4', 4 "-ginseng (benzoyloxyphenyl) methyl, bis- (4', the 4 "-Dimethoxyphenyl) methyl of 3- (imidazoles -1- base), bis- (the 4- first of 1,1- Phenyl) -1'- pyrenylmethy, 9- anthryl, 9- (9- phenyl) dibenzo piperazine mutter base, 9- (side 9- phenyl -10- oxygroup) anthryl, 1,3- benzo dithiolane -2- base, benzisothia oxazolyl S, S- dioxy ion radical, trimethylsilyl (TMS), triethyl group Silylation (TES), tri isopropyl silane base (TIPS), dimethylisopropyl silylation (IPDMS), diethyl isopropyl silylation (DEIPS), dimethyl third hexyl silane base, third butyldimethylsilane base (TBDMS), third butyl diphenyl silylation (TBDPS), trityl silylation, three-paraxylene base silane bases, tri-phenyl-silane base, diphenylmethylsilane base (DPMS), third butyl anisyl silylation (TBMPS), formic acid esters, benzoyl formate, acetic acid esters, chloracetate, two Chloracetate, trichloroacetic esters, trifluoro-acetate, methoxyacetic acid ester, triphenylmethoxy acetic acid esters, phenoxyacetic acid ester, Parachlorophen-oxyacetic acid ester, 3- phenylpropionic acid ester, the side 4- oxygroup valerate (levulinate), 4,4- (stretching ethyl disulfide group) penta Acid esters (levulinic acyl group dithioacetals), pivalate, Buddha's warrior attendant acid esters, crotonates, 4- methoxyl group crotonates, benzoic acid Ester, p-phenyl benzoic acid ester, 2,4,6- trimethylbenzoic acid ester (mesitylene formic acid esters), methyl carbonate, carbonic acid 9- Fluorene base Methyl esters (Fmoc), ethyl carbonate, carbonic acid 2,2,2- trichloro ethyl ester (Troc), 2- (trimethylsilyl) ethyl carbonate (TMSEC), Carbonic acid 2- (phenyl sulfonyl) ethyl ester (Psec), 2- (triphenyl phosphorus base) ethyl carbonate (Peoc), carbonic acid isobutyl ester, carbonic acid second Enester, allyl carbonate, carbonic acid third butyl ester (BOC), p-nitrophenyl carbonate ester, methyl phenyl carbonate, carbonic acid are to methoxybenzene first Ester, carbonic acid 3,4- dimethoxy benzene methyl, carbonic acid ortho-nitrophenyl methyl esters, carbonic acid p-nitrophenyl methyl esters, thiocarbonic acid S- benzene methyl, Carbonic acid 4- ethyoxyl -1- naphthalene ester, dithiocarbonic acids methyl esters, 2- iodo-benzoic acid ester, 4- repeatedly nitrogen base butyrate, 4- nitro -4- first Base valerate, neighbour (two bromomethyls) benzoic ether, 2- formylbenzene sulfonate, 2- (methyl thio methoxyl group) ethyl, 4- (methyl Thiornethoxy group) butyrate, 2- (methyl thio methoxy) benzoic ether, the chloro- 4- methylphenoxyacetate of 2,6- bis-, 2,6- bis- chloro- 4- (1,1,3,3- tetramethyl butyl) phenoxyacetic acid ester, bis- (1,1- dimethyl propyl) phenoxyacetic acids of 2,4- Ester, chlorodiphenyl yl acetate, isobutyrate, monobutane diacid ester, (E) -2- methyl-2-butene acid esters, neighbour (methoxyl group acyl group) benzene Formic acid esters, α-naphthoicacid ester, nitrate, N, N, N', N'- tetramethyl phosphorodiamidate Arrcostab, N- phenylcarbamic acid alkyl Ester, borate, dimethyl phosphino- sulfinyl, dinitrophenyl group sulfinic acid alkyl ester, sulfuric ester, methane sulfonate (methylsulphur Acid esters), benzyl sulphonic acid ester and tosylate (Ts).

Also it has to be noticed that unless the context clearly determines otherwise, otherwise as herein and in appended claims In it is used, singular " one (a) ", " one (an) " and " (the) " include most and refer to object.Equally, term " one (a) " (or " one (an) "), " one or more " and "at least one" are used interchangeably herein.It should also be noted that term "comprising", " packet Include " and " having " be used interchangeably.

Unless otherwise specified, by the molecular biology used within the scope of the technical ability for belonging to technique, microbiology, Recombinant DNA and immunology well-known technique implement the present invention.These technologies are sufficiently explained in the literature.See, for example, Molecular Cloning A Laboratory Manual, second edition, Sambrook, Fritsch and Maniatis compile (Cold Spring Harbor Laboratory Press,1989)；DNA Cloning, I and II volumes (D.N.Glover is compiled, 1985)； Culture Of Animal Cells(R.I.Freshney,Alan R.Liss,Inc.,1987)；Immobilized Cells And Enzymes(IRL Press,1986)；B.Perbal,A Practical Guide To Molecular Cloning (1984)；Paper Methods In Enzymology (Academic Press, Inc., N.Y.)；Gene Transfer (J.H.Miller and M.P.Calos are compiled Vectors For Mammalian Cells, 1987, Cold Spring Harbor Laboratory)；Methods In Enzymology, volume 154 and volume 155 (Wu et al. volume), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker are compiled, Academic Press, London, 1987)；Antibodies:A Laboratory Manual,Harlow and Lane s(Cold Spring Harbor Laboratory Press,1988)；And Handbook Of Experimental Immunology, I-IV volume (D.M.Weir and C.C.Blackwell are compiled, 1986).

As used herein, term " glycan " refers to polysaccharide or oligosaccharides.Glycan is herein also for referring to glycoconjugate Carbohydrate portions, such as glycoprotein, glycolipid, glycopeptide, glycoprotein group, peptide glycan, lipopolysaccharides or proteoglycans.Glycan is logical Often only formed by the O-glycosides between monosaccharide is bonded.For example, cellulose is made of β-Isosorbide-5-Nitrae-connecting-type D-Glucose Glycan (or more particularly, glucan), and chitin is poly- to be made of β-Isosorbide-5-Nitrae-connecting-type N- acetyl group-d-glucosamine Sugar.Glycan can be the homopolymer or heteropolymer of monosaccharide residue, and can be linear or branch.It can find that glycan is connected to albumen Matter, such as glycoprotein and proteoglycans.It is typically found on cell outer surface.O connecting-type and N connecting-type glycan are in eucaryote In it is very common, and (however uncommon) can be found in prokaryotes.It was found that N connecting-type glycan is connected to the day in sequence The R race nitrogen (N) of winter amide.Sequence is Asn-X-Ser or Asn-X-Thr sequence, and wherein X is any ammonia in addition to nut sugar Base acid.

As used herein, term " antigen " is defined as that any substance of immune response can be induced.

As used herein, term " immunogenicity " refers to the ability of immunogene, antigen or boosting vaccine immune response.

As used herein, term " CD1d " refers to glycoprotein expressed on various human antigen-presenting cells surfaces A member of CD1 (differentiation gather together 1) family.The lipidantigen active natural killer T cell that CD1d is presented.CD1d has anti-for glycolipid Original is incorporated into deeper antigen binding groove therein.Expressed CD1d molecule is combinable in Dendritic Cells and presents glycolipid, Including α-GalCer analog, such as C34.

As used herein, term " epitope " is defined as a part of antigen molecule, this part contacts antibody or T The antigen binding site of cell receptor.

As used herein, term " vaccine " refers to the preparation containing antigen, (is killed or is subtracted by complete genic organisms Poison) or these organisms component (such as protein, peptide or polysaccharide) composition, for assigning for disease caused by these organisms The immunity of disease.Vaccine preparation can be natural, synthesis or pass through recombinant DNA technology acquisition.

As used herein, term " antigentic specificity " refers to a kind of characteristic of cell mass, makes specific antigen or anti- The supply of former segment causes specific cell to be proliferated.

As used herein, term " specific binding " refers in conjunction with to the interaction between (such as antibody and antigen). In various situations, specific binding can be presented as about 10^-6Mole/litre, about 10^-7Mole/litre or about 10^-8Mole/litre Or less than 10^-8Mole/litre affinity constant.

As used herein, term carbohydrase at least partly refers to the enzyme in ball series of biologic synthesis path；Illustrative carbohydrase packet Include α -1,4- galactosyltransferase；β -1,4-N- acetyl galactose based transferase-I；Or β -1,3- galactosyltransferase-V.

As used herein, term " ball series of paths " includes biosynthesis described in Fig. 1 and enzymatic path.

" separation " antibody is the antibody for having identified and having separated and/or recycling from the component of its natural surroundings.Its natural surroundings Groups of contaminants be divided into the substance of the research that can interfere antibody, diagnosis or therapeutical uses, and may include enzyme, hormone and other albumen Matter or nonproteinaceous solute.In one embodiment, antibody will purify (1) to more than 95wt%, such as example, by the auspicious method in Lip river (Lowry method) is measured, and in some embodiments more than 99wt%；(2) to be enough by using such as rotary cup survey Sequence instrument obtains the degree of at least 15 residues of N-terminal or internal amino acid sequence；Or (3), to homogeneous, this is by using example Such as library Maas blue (Coomassie blue) or silver staining agent, progress SDS-PAGE reaches under reduction or non reducing conditions.Through Separation antibody includes the antibody in situ being present in recombinant cell, because at least one of natural surroundings of antibody component will not In the presence of.However, in general, will be prepared by least one purification step through separation antibody.

The single binding site collocation object in connection that " binding affinity " generally refers to molecule (such as antibody) is (such as anti- It is former) between noncovalent interaction power summation.Unless otherwise specified, as used herein, " binding affinity " refers to Reflection combines the intrinsic binding affinity to interact to 1:1 between (such as antibody and antigen) member.Molecule X arranges in pairs or groups to it object The affinity of Y can generally be indicated by dissociation constant (Kd).It can be (including described herein by common method known in the art Method) measure affinity.Low-affinity antibody generally slowly combines antigen and tends to easy dissociation, and high-affinity antibody It is general comparatively fast to combine antigen and tend to long period holding bonding state.A variety of measurement binding affinities known in the art Method, any one of them purpose for use in the present invention.Particular illustrative embodiment is described below.

" antibody fragment " only includes a part of complete antibody, wherein the part retains with that part when it has been present in The normally at least function in relevant function, and largely or entirely function when in whole antibody.In one embodiment, antibody Segment includes the antigen binding site of complete antibody and therefore retains the ability for combining antigen.In another embodiment, antibody piece Section (such as including the region Fc person) retains at least one biology usually relevant to the region Fc when being present in complete antibody Function, such as FcRn combine, antibody half life is adjusted, Antibody -dependent cell cytotoxicity kills function and complement combines.? In one embodiment, antibody fragment is the univalent antibody with the vivo half-life for being substantially similar to complete antibody.Citing For, the antibody fragment may include the antigen binding arm for being connected to Fc sequence, can assign to the segment in vivo steady It is qualitative.

The monoclonal antibody of this paper is specific including " chimeric " antibody, a portion heavy chain and/or light chain and derived from specific object Kind or the corresponding sequence that belongs in the antibody of specific antibodies type or subclass it is identical or homologous, and its remaining part of (these) chain Point with derived from another species or to belong to corresponding sequence in the antibody of another antibody type or subclass identical or homologous；And these The segment of antibody, as long as it shows required biological activity (U.S. Patent No. 4,816,567；And Morrison etc. People, Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).

Non-human (such as Muridae) antibody of " humanization " form is to contain the minimum obtained from non-human immunoglobulin The chimeric antibody of sequence.In one embodiment, humanization antibody is human immunoglobulin (receptor antibody), wherein will be obtained from The residue of the hypervariable region of recipient, which is replaced into, is obtained from that (donor resists with specificity, the non-human kind of affinity and/or capacity is wanted Body) (such as mouse, rat, rabbit or non-human primate) hypervariable region residue.In some cases, human immunity ball Framework region (FR) residue of albumen is replaced through corresponding non-human residues.In addition, humanization antibody may include receptor antibody or confession The residue being had no in person's antibody.These modifications are carried out further to improve antibody efficiency.In general, humanization antibody will include Substantially all at least one and usual two variable domains, wherein all or substantially all hypervariable loops correspond to inhuman para-immunity The hypervariable loop of globulin, and the FR that all or substantially all FR are human immunoglobulin sequence.Humanized antibodies are optionally also To include at least part constant region for immunoglobulin (Fc), usually the constant region of human immunoglobulin.For more detailed information, join See Jones et al., Nature 321:522-525 (1986)；Riechmann et al., Nature 332:323-329 (1988)； And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Also cited referring to following commentary paper and wherein Bibliography: Vaswani and Hamilton, Ann.Allergy, Asthma&Immunol.1:105-115 (1998)；Harris, Biochem.Soc.Transactions 23:1035-1038(1995)；Hurle and Gross, Curr.Op.Biotech.5: 428-433(1994)。

The antibody of the bioactivity for the antigen that " blocking " antibody or " antagonist " antibody are combined by inhibition or reduction.It is certain Blocking antibody or antagonist antibodies substantially or completely inhibit the bioactivity of antigen.

" agonist antibody " is by a kind of at least one functional activity for simulating the polypeptide studied as used herein Antibody.

" illness " is any symptom that can be improved through Antybody therapy of the invention.This includes chronic and acute disease or disease Disease, including making mammal be susceptible to suffer from they's pathology patient's condition of the illness.The non-limiting example of illness treated herein includes Cancer.

Term " cell proliferative disorder " and " proliferative disorders " refer to relevant to abnormal cell proliferation to a certain degree Illness.In one embodiment, cell proliferative diseases are cancer.

" tumour " refers to the growth and proliferation of all neoplastic cells (pernicious or benign) as used herein, and all Before cancer and cancer cell and tissue.As recited herein, term " cancer " " canceration ", " cell proliferative disorders ", " proliferative diseases Disease " and " tumour " are not mutually exclusive.

Term " cancer " and " carcinous " refer to or describe in the mammalian body usually with not modulated cell growth/proliferation The physiology patient's condition being characterized.The example of cancer includes but is not limited to carcinoma, lymthoma (such as hodgkin's lymphomas and Fei Huoqi Golden lymphomas), enblastoma, sarcoma and leukaemia.The more specific examples of these cancers include squamous cell carcinoma, cellule lung Cancer, non-small cell lung cancer, the gland cancer of lung, the carcinoma squamosum of lung, peritoneal cancer, hepatocellular carcinoma, human primary gastrointestinal cancers, cancer of pancreas, spongioblast Tumor, cervix cancer, oophoroma, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, carcinoma of endometrium or uterus Cancer, salivary-gland carcinoma, kidney, liver cancer, prostate cancer, carcinoma of vulva, thyroid cancer, liver cancer, leukaemia and other lymphadenias Venereal disease disease and various types of head and neck cancers.

Term " ball series associated disease " refers to or Expressive Features are typically or path function is caused to play or present hair Raw abnormal illness.The example of such illness includes but is not limited to hyperproliferative disease, including cancer.

The example of immunodeficiency disease group includes but is not limited to that ataxia-telangiectasia, leucocyte stick together deficiency disease Hou Qun, lymphopenia, dysgammaglobulinemia disease, human immunodeficiency virus or δ retroviral infection, common change Specific immunological shortage, Reconstruction in Sever Combined Immunodeciency, macrophage sterilizing function exception, agamaglobulinemia, Di Qiaozhi disease Group (DiGeorge syndrome) and the Ritchie Wescott-A De syndrome (Wiskott-Aldrich syndrome).Allergy The example of disease includes but is not limited to allergy, asthma, dermatitis, morbilli, allergy, Wei Sileshi syndrome (Wissler's ) and thrombocytopenic purpura syndrome.

It " treats " as used herein and refers to that the clinic for the natural process for attempting to change individual treated or cell is dry It in advance, and can be for progress during the purpose or clinicopathologia of prevention.Ideal therapeutic effect include prevent seizure of disease or Recurrence, prevents or slows down inflammation and/or tissue/organ at any direct or indirect pathological consequences for mitigating symptom, reducing disease Injury reduces disease travel speed, improves or slow down morbid state and remission or prognosis improvement.In some embodiments, The development of disease or illness is postponed using antibody of the invention.

" individual (individual) " or " individual (subject) " is vertebrate.In certain embodiments, vertebrate For mammal.Mammal includes but is not limited to farming animals (such as ox), sport animals, pet (such as cat, dog and horse), spirit Long class animal, mouse and rat.In certain embodiments, vertebrate is the mankind.

" mammal " for therapeutic purposes refers to any animal for being classified as mammal, including the mankind, raise and train it is dynamic Object and agro-farming animal and zoo, movement or pet animals, dog, horse, cat, cow etc..In certain embodiments, it feeds Newborn animal is the mankind.

" effective quantity " refers to dosage and time quantum necessary to effectively reaching required treatment or prevention result.

" therapeutically effective amount " of substances/molecules of the invention can become according to such as following factor: individual morbid state, Age, gender and weight and the substances/molecules induce institute's ability to be reacted in individual.Therapeutically effective amount is also one Kind treatment beneficial effect is more than any toxicity of the substances/molecules or the amount of ill-effect." prevention effective dose " refers to effectively Reach dosage necessary to required prevention result and time quantum.Due to preventive dose be before illness or illness early stage It is used in body, therefore prevention effective dose usually (but not necessarily) is less than therapeutically effective amount.

The term as used herein " cytotoxic agent " refer to it is a kind of inhibition or prevent cell function and/or cause cytoclasis Substance.The term is intended to include radioactive isotope (such as At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、 P³²And the radioactive isotope of Lu)；Chemotherapeutant, such as methotrexate, adriamycin (adriamicin), Vinca alkaloids class (vinca alkaloids) (vincristine (vincristine), vinblastine (vinblastine), Etoposide (etoposide), adriamycin (doxorubicin), melphalan (melphalan), mitomycin C (mitomycin C), benzene fourth Sour mustargen (chlorambucil), daunomycin (daunorubicin) or other inserting agents, enzyme and its segment (such as nucleic acid point Solution enzyme, antibiotic and toxin, such as small molecule toxins of bacterium, fungi, plant or animal origin or enzymatic activity toxin, including Its segment and/or variant) and various antitumor agents or anticancer agent disclosed below.Other cytotoxic agents are hereinafter retouched It states.Tumor-killing agent can be such that tumour cell damages.

" chemotherapeutant " is the compound that can be used for treating cancer.The example of chemotherapeutant includes: alkylating agent, such as Thiotepa (thiotepa) andCyclophosphamide；Alkyl sulfonates, such as busulfan (busulfan), English Third Shu Fan (improsulfan) and piposulfan (piposulfan)；Aziridines, such as benzene azoles DOPA (benzodopa), card Bar quinone (carboquone), rice spy DOPA (meturedopa) and outstanding benefit bar (uredopa)；It stretches ethylimido class and methyl is close Amine, including hemel (altretamine), three stretch ethyl melamine (triethylenemelamine), three stretch ethyl phosphinylidyne Amine (trietylenephosphoramide), three stretch ethylenebis dithiocarbamate phosphamide (triethiylenethiophosphoramide) And front three melamine (trimethylolomelamine)；Acetogenin class (acetogenins) (especially bullatacin (bullatacin) and bullatacinone (bullatacinone))；Δ -9- tetrahydrocannabinol (delta-9- Tetrahydrocannabinol) (Dronabinol (dronabinol,))；β-lapachone (beta- lapachone)；Lapachol (lapachol)；Colchicin (colchicine)；Betulinic acid (betulinic acid)；Camplotheca acuminata Alkali (camptothecin) (including synthetic analogues topotecan (topotecan)CPT-11 (Yi Nuo For health (irinotecan；), acetyl group camptothecine (acetylcamptothecin), the general Lay of scott sting (scopolectin) and 9-aminocamptothecin)；Bryostatin (bryostatin)；This tower of Cali stings (callystatin)；CC- 1065 (including its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin synthetic analogues)；Foot Leaf grass toxin (podophyllotoxin)；Podophyllinic acid (podophyllinic acid)；Teniposide (teniposide)； Cryptophycin (cryptophycin) (especially cryptophycin 1 and cryptophycin 8)；Aplysiatoxin (dolastatin)； More Ka meter Xin (duocarmycin) (including synthetic analogues, KW-2189 and CB1-TM1)；Ai Liusu (eleutherobin)； Disk Ke Sita stings (pancratistatin)；The spit of fland (sarcodictyin) that sand is examined；Spongistatin (spongistatin)；Mustargen Class, such as Chlorambucil, Chlornaphazine (chlornaphazine), cyclophosphamide, estramustine (estramustine), different ring phosphorus Amide, mechlorethamine, Mechlorethamine hydrochloride oxide (mechlorethamine oxide Hydrochloride), melphalan, novoembichin (novembichin), phenesterine (phenesterine), Song Long Benzene mustard (prednimustine), trofosfamide (trofosfamide), uracil mastard (uracil mustard)；Nitre Base urea, such as carmustine (carmustine), chloramphenicol (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and La Ningsiting (ranimnustine)；Antibiotic, such as enediyne antibiotic (such as calicheamycin (calicheamicin), especially thorn spore are mould Plain γ 1 and calicheamycin ω 1 (see, for example, Agnew, Chem Intl.Ed.Engl., 33:183-186 (1994))；Da meter Xin Including Da meter Xin A (dynemicin),；Ai Sipa meter Xin (esperamicin)；And neoearcinostain (neocarzinostatin) chromophore and related chromoprotein enediyne antibiotic chromophore), Peter Krass rice it is pungent (aclacinomysins), D actinomycin D (actinomycin), Austria Si La meter Xin (authramycin), diazonium silk amino acid (azaserine), bleomycin (bleomycin), act-C (cactinomycin), OK a karaoke club than pungent (carabicin), Carminomycin (carminomycin), cardinophyllin (carzinophilin), chromium pungent (chromomycinis), actinomyces Plain (dactinomycin), daunomycin, Detorubicin (detorubicin), the just white amino acid of 6- diazo -5- side oxygen-L-,Adriamycin (including morpholine simultaneously-adriamycin (morpholino-doxorubicin), cyano morpholine simultaneously- Adriamycin (cyanomorpholino-doxorubicin), 2- pyrrolo--adriamycin and deoxy doxorubicin (deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) (such as mitomycin C), mould Phenolic acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycins), Peplomycin (peplomycin), sprinkle non-mycin (potfiromycin), puromycin (puromycin), Kui that mycin (quelamycin), Rodorubicin (rodorubicin), streptozotocin (streptozocin), kills tulase at streptonigrin (streptonigrin) Element (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin)；It is anti- Metabolin, such as methotrexate and 5 FU 5 fluorouracil (5-fluorouracil) (5-FU)；Folacin, such as Dai Nationality's promise spy purine (denopterin), methotrexate, pteropterin (pteropterin), Trimetrexate (trimetrexate)；Purine analogue, Such as fludarabine (fludarabine), Ismipur (6-mercaptopurine), thiophene miaow purine (thiamiprine), Thioguanine (thioguanine)；Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), azauridine (6-azauridine), Carmofur (carmofur), cytarabine (cytarabine), double de- Oxygen uridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), fluorine urinate core Glycosides (floxuridine)；Male sex hormone, such as clausterone (calusterone), Masterone (dromostanolone propionate), epithioandrostanol (epitiostanol), Mepitiostane (mepitiostane), in testis Ester (testolactone)；Anti- adrenal gland agent, such as aminophenethylphenidone (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane)；Folic acid supplement, such as Fu Luolin acid (frolinic acid)；In vinegar Portugal Ester (aceglatone)；Aldophosphamide glucoside (aldophosphamide glycoside)；Aminolevulinic acid (ALA) (aminolevulinic acid)；Erie Lu draws (eniluracil)；Amsacrine (amsacrine)；Times Si Tabu (bestrabucil)；Bisantrene (bisantrene)；Angstrom reach Qu Ke (edatraxate)；It obtains and not cuts down amine (defofamine)；Autumn Narcissamine (demecolcine)；Diaziquone (diaziquone)；James Ivory dissipates (elfornithine)；Elliptinium Acetate (elliptinium acetate)；Epothilones (epothilone)；Etoglucid (etoglucid)；Gallium nitrate (gallium nitrate)；Hydroxycarbamide (hydroxyurea)；Lentinan (lentinan)；Luo Nidaning (lonidainine)；CHROMATOGRAPHIC FRACTIONATION AND MASS (maytansinoids), such as maytansine (maytansine) and amine Sha Tuoxin (ansamitocins)；Methyl-GAG (mitoguazone)；Mitoxantrone (mitoxantrone)；Not than Bodhidharma (mopidanmol)；Nitre La Weilin (nitraerine)；Pentostatin (pentostatin)；Fan Naming (phenamet)；Pirarubicin (pirarubicin)；Lip river Rope anthraquinone (losoxantrone)；2- ethylhydrazide (2-ethylhydrazide)；Procarbazine (procarbazine)；Polysaccharide compound (JHS Natural Products, Eugene, OR)；Tetrahydroform (razoxane)；Head mold rhzomorph (rhizoxin)；Sizofiran (sizofiran)；Spiral shell germanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2.2'-2 "-ethylaluminum amine；Trichothecenes toxin (trichothecenes) (especially T-2 toxin, Fu Nakulin A (verracurin A), Roridine A (roridin A) and amine The last of the ten Heavenly stems stings (anguidine))；Urethane (urethan)；Desacetyl vinblastine amide (vindesine) Dacarbazine (dacarbazine)；Mannomustin (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；First support is pungent (gacytosine)；Cytarabine (arabinoside) (" Ara-C ")；Thiotepa (thiotepa)；Taxol (taxoids), Such asPaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.)； ABRAXANETM designs nanoparticle composite (American without the albumin of Cremophor- Paclitaxel Pharmaceutical Partners, Schaumberg, Illinois) andDocetaxel (doxetaxel)(-Poulenc Rorer,Antony,France)；Crow south cloth (chloranbucil)；Ji Xita Shore (gemcitabine)6- thioguanine (6-thioguanine)；Purinethol (mercaptopurine)；Methotrexate；Platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin)；It is long Spring flower alkali (vinblastine)Platinum；Etoposide (VP-16)；Ifosfamide；Mitoxantrone (mitoxantrone)；Vincristine (vincristine)Oxaliplatin (oxaliplatin)；Lu examines Fu Wen (leucovovin)；Vinorelbine (vinorelbine)Nuo Fantelong (novantrone)； Edatrexate (edatrexate)；Daunomycin；Aminopterin；Ibandronate (ibandronate)；Topoisomerase inhibits Agent RFS 2000；Difluoromethyl bird amino acid (difluorometlhylornithine) (DMFO)；Retinoid class, such as view are yellow Sour (retinoic acid)；Capecitabine (capecitabine)The medicine of any of the above therapeutic agent Acceptable salt, acid or derivative on；And the combination of both the above or two or more therapeutic agents, such as CHOP (ring phosphinylidyne Amine, adriamycin, vincristine and prednisolone (prednisolone) mixing treatment abbreviation) and FOLFOX (oxaliplatin (oxaliplatin) abbreviation of scheme is treated in combination in (ELOXATINTM) and 5-FU and Lu Kaofuwen).

Unless defined otherwise, otherwise all technologies used herein and scientific term have and are generally familiar with the present invention The identical meaning of the meaning that affiliated operator is generally understood.Although similar or be equivalent to they's method described herein And any method and material of material can be used for practicing or testing in the present invention, but preferred method and material will now be described.Herein Specifically mentioned all publication and patent are incorporated herein by reference for all purposes, including describe and disclose public affairs It opens in case and reports, chemicals, cell strain, carrier, animal, instrument, statistical analysis and method that the present invention uses can be combined. Cited all bibliography are considered as the instruction of the level of skill in technique in this specification.It should not be construed as holding herein Recognize the such disclosure of the invention had no right prior to prior inventions.

In an aspect, the present invention is based on following surprising discovery: phase specificity embryonal antigen (SSEA3 and SSEA4) The stabilization IgG antibody reaction for specifically identifying SSEA3 and SSEA4 respectively can be induced through certain base group modifications.

In some instances, the modification of SSEA3 includes the fluorine on one or more positions of the glucose of SSEA3, repeatedly nitrogen base Or O- phenyl.In some instances, the fluorine of SSEA3 modified on one or more positions comprising non-reducing end galactolipin, repeatedly nitrogen Base or O- phenyl.In some instances, the fluorine on one or more positions for modifying the glucose comprising SSEA4 of SSEA4, repeatedly nitrogen Base or O- phenyl.In some instances, the modification of SSEA4 includes the fluorine on one or more positions of sialic acid residues, repeatedly nitrogen base Or O- phenyl.

In certain aspects, the present invention provides SSEA3 the and SSEA4 class for having modification in reducing end and/or non-reducing end Like object.Compared to primary SSEA3 and SSEA4, such SSEA3 and SSEA4 analog can induce stronger immune response and (such as lure IgG antibody of the guide pin to SSEA3 and/or SSEA4).Immunogenic composition comprising such non-natural glycan moiety is induced Antibody can mediate the cytotoxicity of the complement dependent cellular for tumour cell.

Compound

Therefore, the present invention also shows the Noval compounds being made of modified Carbohydrate Antigens (SSEA3 and SSEA4) Object, glycan conjugate and its immunogenic composition and vaccine comprising such compound.

In an aspect, the present invention provides formula (I) compound:

Or its salt,

Wherein:

X₁For-OR or-SR, wherein R is hydrogen, oxygen or sulfur protecting group, the C1-10 alkyl that is optionally substituted, optionally passes through Substituted aryl, the acyl group being optionally substituted or the acylimino being optionally substituted；

R¹、R²、R³、R⁴、R⁵、R⁶And each example of L is independently selected from hydrogen, halogen, the optionally alkyl, optionally that is substituted The alkenyl that is substituted, the alkynyl being optionally substituted, the heterocycle being optionally substituted, the aryl ,-N being optionally substituted₃、- NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S (O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, optionally pass through Substituted alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, optionally pass through Substituted alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

Its restrictive condition is that compound does not have following formula:

In certain embodiments, X₁In α configuration.In certain embodiments, X₁In β configuration.

In some embodiments, X₁For-OR^A.In some embodiments, X₁For-OH.In some embodiments, X₁For-O- (protecting group).In some embodiments, X₁For-OR^A, wherein R^AFor the C1-10 alkyl being unsubstituted.In some embodiments, X₁ For-OR^A, wherein R^AFor the C1-10 alkyl being substituted.In some embodiments, X₁For-OR^A, wherein R^AFor the virtue being unsubstituted Base.In some embodiments, X₁For-OR^A, wherein R^AFor the aryl being substituted.In some embodiments, X₁For-OR^A, wherein R^A For the acyl group being unsubstituted.In some embodiments, X₁For-OR^A, wherein R^AFor the acyl group being substituted.In some embodiments, X₁For-OR^A, wherein R^AFor the acylimino being unsubstituted.In some embodiments, X₁For-OR^A, wherein R^AFor the acyl being substituted Imino group.

In some embodiments, X₁For-SR^A.In some embodiments, X₁For-SH.In some embodiments, X₁For-S- (protecting group).In some embodiments, X₁For-SR^A, wherein R^AFor the C1-10 alkyl being unsubstituted.In some embodiments, X₁ For-SR^A, wherein R^AFor the C1-10 alkyl being substituted.In certain embodiments, X₁For-SCH₃.In some embodiments, X₁For- SR^A, wherein R^AFor the aryl being unsubstituted.In some embodiments, X₁For-SR^A, wherein R^AFor the aryl being substituted.Some In embodiment, X₁For-SR^A, wherein R^AFor the acyl group being unsubstituted.In some embodiments, X₁For-SR^A, wherein R^ATo be substituted Acyl group.In some embodiments, X₁For-SR^A, wherein R^AFor the acylimino being unsubstituted.In some embodiments, X₁For- SR^A, wherein R^AFor the acylimino being substituted.

In some embodiments, X₁For C1-10 alkoxy.In some embodiments, X₁For C1-3 alkoxy.

In some embodiments, X₁Selected from the group being made up of: α-butylthiomethyl, β-butylthiomethyl, α-sulfenyl toluene Base, β-sulfenyl tolyl, α-third butyl diphenyl silylation oxygroup, β-third butyl diphenyl silylation oxygroup and α-methoxy Base.

In some embodiments, R¹For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R¹For-N₃.In certain embodiments, R¹For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R¹For-NH₂.In certain embodiments, R¹For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R¹ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R¹Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R¹ For-NH (Cbz).In certain embodiments, R¹For-NH (Fmoc).In certain embodiments, R¹For-NHC (O) CCl₃.Certain In embodiment, R¹For-NHC (O) CH₃.In certain embodiments, R¹For-N (C (O) CH₃)₂。

In some embodiments, R²For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R²For-N₃.In certain embodiments, R²For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R²For-NH₂.In certain embodiments, R²For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R² For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R²Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R² For-NH (Cbz).In certain embodiments, R²For-NH (Fmoc).In certain embodiments, R²For-NHC (O) CCl₃.Certain In embodiment, R²For-NHC (O) CH₃.In certain embodiments, R²For-N (C (O) CH₃)₂。

In some embodiments, R³For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R³For-N₃.In certain embodiments, R³For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R³For-NH₂.In certain embodiments, R³For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R³ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R³Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R³ For-NH (Cbz).In certain embodiments, R³For-NH (Fmoc).In certain embodiments, R³For-NHC (O) CCl₃.Certain In embodiment, R³For-NHC (O) CH₃.In certain embodiments, R³For-N (C (O) CH₃)₂。

In some embodiments, R⁴For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R⁴For-N₃.In certain embodiments, R⁴For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R⁴For-NH₂.In certain embodiments, R⁴For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R⁴ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R⁴Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R⁴ For-NH (Cbz).In certain embodiments, R⁴For-NH (Fmoc).In certain embodiments, R⁴For-NHC (O) CCl₃.Certain In embodiment, R⁴For-NHC (O) CH₃.In certain embodiments, R⁴For-N (C (O) CH₃)₂。

In some embodiments, R⁵For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R⁵For-N₃.In certain embodiments, R⁵For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R⁵For-NH₂.In certain embodiments, R⁵For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R⁵ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R⁵Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R⁵ For-NH (Cbz).In certain embodiments, R⁵For-NH (Fmoc).In certain embodiments, R⁵For-NHC (O) CCl₃.Certain In embodiment, R⁵For-NHC (O) CH₃.In certain embodiments, R⁵For-N (C (O) CH₃)₂。

In some embodiments, R⁶For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R⁶For-N₃.In certain embodiments, R⁶For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R⁶For-NH₂.In certain embodiments, R⁶For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R⁶ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R⁶Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R⁶ For-NH (Cbz).In certain embodiments, R⁶For-NH (Fmoc).In certain embodiments, R⁶For-NHC (O) CCl₃.Certain In embodiment, R⁶For-NHC (O) CH₃.In certain embodiments, R⁶For-N (C (O) CH₃)₂。

In some embodiments, R¹、R²And R³It is identical.In some embodiments, R¹、R²And R³For-OH.In some embodiments In, R⁴、R⁵And R⁶It is identical.In some embodiments, R⁴、R⁵And R⁶For-OH.

In certain embodiments, L is-OH.

In certain embodiments, L is-OH and R¹For-N₃.In certain embodiments, L is-OH, R¹For-N₃, and R²、R³、 R⁴、R⁵And R⁶Each example be-OH.

In certain embodiments, L is-OH and R²For-N₃.In certain embodiments, L is-OH, R²For-N₃And R¹、R³、R⁴、 R⁵And R⁶Each example be-OH.

In certain embodiments, L is-OH and R³For-N₃.In certain embodiments, L is-OH, R³For-N₃, and R¹、R²、 R⁴、R⁵And R⁶Each example be-OH.

In certain embodiments, L is-OH and R⁴For-N₃.In certain embodiments, L is-OH, R⁴For-N₃, and R¹、R²、 R³、R⁵And R⁶Each example be-OH.

In certain embodiments, L is-OH and R⁵For-N₃.In certain embodiments, L is-OH, R⁵For-N₃, and R¹、R²、 R³、R⁴And R⁶Each example be-OH.

In certain embodiments, L is-OH and R⁶For-N₃.In certain embodiments, L is-OH, R⁶For-N₃, and R¹、R²、 R³、R⁴And R⁵Each example be-OH.

In certain embodiments, R¹、R²、R³、R⁴、R⁵、R⁶And each example of L is-F.In certain embodiments, R¹For-F. In certain embodiments, R²For-F.In certain embodiments, R³For-F.In certain embodiments, R⁴For-F.In some embodiments In, R⁵For-F.In certain embodiments, R⁶For-F.In certain embodiments, L is-F.

In certain embodiments, L has a structure that

Wherein:

R⁸、R⁹、R¹⁰And R¹¹Each example independently selected from hydrogen, halogen, optionally be substituted alkyl, be optionally substituted Alkenyl, the alkynyl, the heterocycle being optionally substituted, the aryl ,-N that are optionally substituted that are optionally substituted₃、-NO₂、-N (R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

R_NSelected from-N₃、-NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C (O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, optionally pass through Substituted alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, optionally pass through Substituted alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted.

In some embodiments, compound has formula (II)

Wherein: R¹、R²、R³、R⁸、R⁹、R¹⁰、R¹¹And R_NAnd X₁As described herein, and

Its restrictive condition is that compound does not have following formula:

In some embodiments, R⁸For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R⁸For-N₃.In certain embodiments, R⁸For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R⁸For-NH₂.In certain embodiments, R⁸For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R⁸ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R⁸Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R⁸ For-NH (Cbz).In certain embodiments, R⁸For-NH (Fmoc).In certain embodiments, R⁸For-NHC (O) CCl₃.Certain In embodiment, R⁸For-NHC (O) CH₃.In certain embodiments, R⁸For-N (C (O) CH₃)₂。

In some embodiments, R⁹For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R⁹For-N₃.In certain embodiments, R⁹For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R⁹For-NH₂.In certain embodiments, R⁹For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R⁹ For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R⁹Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R⁹ For-NH (Cbz).In certain embodiments, R⁹For-NH (Fmoc).In certain embodiments, R⁹For-NHC (O) CCl₃.Certain In embodiment, R⁹For-NHC (O) CH₃.In certain embodiments, R⁹For-N (C (O) CH₃)₂。

In some embodiments, R¹⁰For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R¹⁰For-N₃.In certain embodiments, R¹⁰For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R¹⁰For-NH₂.In certain embodiments, R¹⁰For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R¹⁰For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R¹⁰Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R¹⁰For-NH (Cbz).In certain embodiments, R¹⁰For-NH (Fmoc).In certain embodiments, R¹⁰For-NHC (O) CCl₃.? In some embodiments, R¹⁰For-NHC (O) CH₃.In certain embodiments, R¹⁰For-N (C (O) CH3)₂。

In some embodiments, R¹¹For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R¹¹For-N₃.In certain embodiments, R¹¹For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R¹¹For-NH₂.In certain embodiments, R¹¹For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R¹¹For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R¹¹Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R¹¹For-NH (Cbz).In certain embodiments, R¹¹For-NH (Fmoc).In certain embodiments, R¹¹For-NHC (O) CCl₃.? In some embodiments, R¹¹For-NHC (O) CH₃.In certain embodiments, R¹¹For-N (C (O) CH₃)₂。

In some embodiments, R¹²For-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R¹²For-N₃.In certain embodiments, R¹²For-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R¹²For-NH₂.In certain embodiments, R¹²For-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R¹²For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R¹²Selected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R¹²For-NH (Cbz).In certain embodiments, R¹²For-NH (Fmoc).In certain embodiments, R¹²For-NHC (O) CCl₃.? In some embodiments, R¹²For-NHC (O) CH₃.In certain embodiments, R¹²For-N (C (O) CH₃)₂。

In some embodiments, R_NFor-N₃Or-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.In certain realities It applies in example, R_NFor-N₃.In certain embodiments, R_NFor-N (RW)₂, wherein each RW independently is hydrogen or nitrogen-protecting group.Certain In embodiment, R_NFor-NH₂.In certain embodiments, R_NFor-NHRW, wherein RW is nitrogen-protecting group.In certain embodiments, R_N For-N (RW)₂, wherein each RW is nitrogen-protecting group.In certain embodiments, R_NSelected from the group being made up of :-N₃、-NH (Cbz)、-NH(Boc)、-NH(Fmoc)、-NHC(O)CCl₃、-NHC(O)CH₃And-N (C (O) CH₃)₂.In certain embodiments, R_N For-NH (Cbz).In certain embodiments, R_NFor-NH (Fmoc).In certain embodiments, R_NFor-NHC (O) CCl₃.Certain In embodiment, R_NFor-NHC (O) CH₃.In certain embodiments, R_NFor-N (C (O) CH₃)₂。

Immunogenic composition

In another aspect, the present invention provides a kind of immunogenic composition, and comprising (a) include carrier and one or more are poly- The glycan conjugate of sugar, and optionally existing (b) adjuvant,

Wherein: each of one or more glycan via connector in conjunction with carrier, with formula (III) or (IV):

Wherein X₁、R¹、R²、R³、R⁴、R⁵、R⁶、R⁸、R⁹、R¹⁰、R¹¹, L and R_NAs described herein.

In certain embodiments, connector is Heterobifunctional connector or bifunctional connector.

In certain embodiments, connector is bifunctional p-nitrophenyl connector.

In certain embodiments, connector includes at least one sulphur atom, carboxylate group, acylamino- group, carbamic acid Ester group, carbonate group, thiocarbamate group, thiocarbonate group, sulfide group, maleimide Group, N- hydroxyl maleimide group, or any combination thereof.

In certain embodiments, connector is-L¹-L², wherein L¹For a key ,-O- ,-S- ,-NRL1a- ,-C (=O)-,- NRL1aC (=O)-,-NRL1aC (=O) O- ,-C (=O) NRL1a- ,-OC (=O) NRL1a- ,-SC (=O)-,-C (=O) S- ,-OC (=O)-,-C (=O) O- ,-NRL1aC (=S)-,-C (=S) NRL1a-, trans- CRL1b=CRL1b-, cis- CRL1b =CRL1b- ,-C ≡ C- ,-OC (RL1b) 2- ,-C (RL1b) 2O- ,-NRL1aC (RL1b)₂-、-C(RL1b)₂NRL1a-、-SC (RL1b)₂,-C (RL1b) 2S- ,-S (=O)₂O- ,-OS (=O)₂,-S (=O)₂NRL1a- ,-NRL1aS (=O)₂-；Or view feelings The C1-20 hydrocarbon chain that condition is substituted, optionally wherein one or more carbon units of the hydrocarbon chain through following displacement :-O- ,-S- ,- NRL1a- ,-C (=O)-, NRL1aC (=O)-,-NRL1aC (=O) O- ,-C (=O) NRL1a- ,-OC (=O) NRL1a- ,-SC (=O)-,-C (=O) S- ,-OC (=O)-,-C (=O) O- ,-NRL1aC (=S)-,-C (=S) NRL1a-, trans- CRL1b= CRL1b-, cis- CRL1b=CRL1b- ,-C ≡ C- ,-S (=O)₂O- ,-OS (=O)₂,-S (=O)₂NRL1a- or-NRL1aS (=O)₂, wherein RL1a is that hydrogen, the C1-6 alkyl being optionally substituted or nitrogen-protecting group or RL1a are connect with adjacent carbon atom And the heterocycle being optionally substituted is formed, and the RL1b wherein occurred every time is independently selected from the group being made up of: hydrogen, halogen Element, the alkenyl being optionally substituted, the alkynyl being optionally substituted, is optionally substituted the C1-10 alkyl being optionally substituted Carbocylic radical, the heterocycle being optionally substituted, the aryl being optionally substituted and the heteroaryl or RL1b that are optionally substituted It is connect with adjacent carbons or nitrogen or oxygen atom and forms the carbocyclic ring being optionally substituted or heterocycle or the connection of two RL1b groups and shape At the carbocyclic ring being optionally substituted or the heterocycle being optionally substituted；And L²For carrier and L can be made¹The cross-linking reagent institute of crosslinking Derivative part.

Carrier can be the dendritic of protein, lipid, lipidization protein, virus, peptide or glycopeptide.Certain In embodiment, carrier is the peptide that base is determined comprising T cell antigen.

The carrier protein matter example that can be used in the present invention is tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria Toxin cross reacting material 197 (CRM197), the segment C of TT, keyhole spiral shell blood cyanogen albumen (KLH), bovine serum albumin(BSA) (BSA), 3-protein d, outer membrane protein (OMP) and pneumolysin, diphtheria toxin cross reacting material 197 (CRM197) or its His DT point mutation body, such as CRM176, CRM228, CRM45 (Uchida et al., J.Biol.Chem.218；3838-3844, 1973)；Other mutation described in CRM9, CRM45, CRM102, CRM103 and CRM107 and technique.

In certain embodiments, glycan conjugate has formula (IV-a) or (IV-b):

Wherein m is integer 1 to 40 (including endpoint).

In certain embodiments, m is integer 1 to 30 (including endpoint).Such as this paper general definition, m is integer 1 to 20 (including endpoint).In certain embodiments, 1 m.In certain embodiments, 2 m.In certain embodiments, 4 m.Certain In embodiment, m 6.In certain embodiments, 8 m.In certain embodiments, 10 m.In certain embodiments, 15 m. In certain embodiments, 20 m.In certain embodiments, 30 m.In certain embodiments, 40 m.

In another aspect, the present invention provides the glycan knot comprising at least the two in glycan conjugate as described herein Polymer mixtures.In certain embodiments, the w average value in oligosaccharide mixture is about 1.0 to about 40.0.In some embodiments In, the w average value in oligosaccharide mixture is about 1.0 to 10.0.In certain embodiments, the w average value in oligosaccharide mixture is About 5.7,4.9,2.9,2.8 or 3.1.In certain embodiments, the w average value in oligosaccharide mixture be about 4.9,2.9,2.8 or 3.1。

In certain embodiments, immunogenic composition as described herein includes a effective amount of glycan of the present invention in immunology Conjugate.In certain embodiments, immunogenic composition includes pharmaceutically a effective amount of glycan conjugate of the present invention.

Program as described herein can be used to synthesize and also referring to US20140051127 for the compounds of this invention.

Immunogenic conjugates of the invention may include identical or different SSEA3 and/or SSEA4 analog and/or correlation One or more molecules (such as 1-40,1-20,1-25,1-30 kind) of derivative.For generating other descriptions of glycan conjugate And relative program is described as follows.Also referring to U.S. Patent No. 8,268,969.Its content is incorporated herein by reference.

In certain embodiments, immunogenic composition of the invention may include one or more adjuvants.Suitable adjuvant can wrap Include such as C34,7DW8-5, C17, C23, C-30, alpha-galactosylceramide, Glc-C34, aluminium salt, squalene, MF59 and QS- 21)。

As used herein, term " aluminium adjuvant " refers to the aluminium salt with immunolgical adjuvant activity.In this medicament adsorbent solution Proteantigen and make its Shen Dian；Gained sediment is slow by promoting the antigen being formed by vaccine storage tank at inoculation site On The Drug Release improves vaccine immunogenicity.

As used herein, term " immunologic adjuvant " refers to the enhancing used in conjunction with immunogene or metering needle to immunogene The substance of immune response.α-GalCer analog of the invention is used as adjusting or enhancing the immunologic adjuvant of vaccine effect, leads to The immune system of the patient of stimulation oversaturation vaccine institute administration is to generate more strong reaction to vaccine.In exemplary embodiments, make Use analog C34 as adjuvant.The structure of C34 and other alpha-galactosylceramide analogs and its purposes as adjuvant are taken off It is shown in U.S. Patent No. 7,928,077.

As used herein, term " glycolipid " refers to the lipid of carbohydrate connection, serves as the label of cell recognition.

Glycolipid C34, Glc-C34, C23 and 7DW8-5 have a structure that

Immunogenic composition can further comprise pharmaceutically acceptable excipient.In certain embodiments, herein The immunogenic composition includes pharmaceutically a effective amount of glycan conjugate of the present invention.

In another aspect, the present invention is provided comprising immunogenic composition as described herein and pharmaceutically acceptable The cancer vaccine of excipient.

Cancer vaccine of the invention may include the glycan conjugate of the present invention of single dose or multidose, the combination of its glycan Object mixture or its immunogenic composition.Provided cancer vaccine is applicable to treating cancer or reduces risk of cancer.Cancer Disease vaccine also may include packaged information, describe purposes or prescription information for individual or health-care professional person.Supervisor Structure, such as U.S.'s food and Drug Administration (U.S.Food and Drug Administration；FDA), it may be necessary to this Category information.Cancer vaccine also can optionally include the device of administration compound or composition, such as the injection of parenteral dispensing Device.

Pharmaceutical formulation

Immune composition is in a manner of compatible with dosage form and to have validity, protectiveness and immunogenicity in treatment Measure administration.Dosage individual immunity system synthesis antibody and generates cell depending on individual treated, including for example when necessary The ability of mediated immunity reaction.The correct amount of active constituent needed for dispensing is depending on practitioner's judgement.However, being familiar with this skill Patient is easy to determine suitable dosage range.Initial dispensing and add that beat the suitable scheme of dosage be also variable, but at the beginning of may include Begin to offer medicine, then offer medicine again.Vaccine dose can also become depending on dosing way and according to host's figure.

Immune composition of the invention also can be used in the animal for generating antibody generate can be used for treatment of cancer with The antibody of diagnosis.The method for generating single plant and multi-strain antibody and its segment with animal (such as mouse, rabbit, goat, sheep or horse) It is well known in the art.See, for example, Harlow and Lane, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory,New York.Term " antibody " include entire immunoglobulin molecule and its Segment, such as Fab, F (ab')₂, Fv, scFv (single-chain antibody) and dAb (domain antibodies；Ward et al., (1989) Nature, 341, 544)。

Composition disclosed herein can be read the present invention with those who familiarize themselves with the technology and identifiable other activating agents, Carrier, mediator, excipient or auxiliary agent are included in medical composition together.

Medical composition preferably comprises at least a kind of pharmaceutically acceptable carrier.In such medical composition, this The revealed composition of text forms " reactive compound ", also known as " activating agent ".As used herein, language " can pharmaceutically connect The carrier received " includes solvent, decentralized medium, coating agent, antibacterial agent and the antifungal agent compatible with offeing medicine, isotonic agent and absorption Delayed-action activator and the like.Also supplement reactive compound can be incorporated in composition.Medical composition is formulated to be expected with it Dosing way it is compatible.The example of dosing way includes parenteral, for example, intravenously, intradermal, subcutaneous, oral (such as sucking), Percutaneously (part), through mucous membrane and rectal administration.It may include following for parenteral, intradermal or subcutaneous administration solution or suspension Component: sterile diluent, such as water for injection, physiological saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or Other synthetics；Antibacterial agent, such as benzyl alcohol or methyl p-hydroxybenzoate；Antioxidant, such as ascorbic acid or Asia Sodium bisulfate；Chelating agent, such as ethylenediamine tetra-acetic acid；Buffer, such as acetate, citrate or phosphate；And tension tune Save agent, such as sodium chloride or dextrose.Usable acid or alkali (such as hydrochloric acid or sodium hydroxide) adjust pH.Parenteral preparation can encapsulate In the ampoule made of glass or plastics, disposable injection syringe or multiple dose vials.

Clinical application

The present invention provides glycan conjugate, immunogenic composition or the epidemic disease for being suitable for treating the proliferative disease of individual Seedling, proliferative disease such as cancer (such as lung cancer, colorectal cancer, cancer of pancreas, cancer of bile ducts or carcinoma of endometrium), benign anything superfluous or useless or blood Pipe generates.

Immunogenic composition or vaccine as described herein also can be used for producing in the mankind or animal for generating antibody The raw antibody that can be used for treatment of cancer and diagnosis.In some embodiments, immunogenic composition or vaccine as described herein be also Can be used for that GloboH, SSEA3 and/or SSEA4 antibody is caused to generate.It is produced with animal (such as mouse, rabbit, goat, sheep or horse) The method of raw single plant and multi-strain antibody and its segment is well known in the art.See, for example, Harlow and Lane, (1988) Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,New York.Term is " anti- Body " includes entire immunoglobulin molecule and its segment, such as Fab, F (ab)₂, Fv, scFv (single-chain antibody) and (domain dAb Antibody；Ward et al., (1989) Nature, 341,544).

It provides to contain comprising at least one anti-SSEA3/SSEA4/GloboH antibody or at least one and encodes anti-SSEA3/ The composition of the polynucleotide of the sequence of SSEA4/GloboH antibody.In certain embodiments, composition can be medical composition. As used herein, composition includes that one or more are bound to the antibody and/or one of one or more SSEA3/SSEA4/GloboH Or it is a variety of containing encode one or more be bound to the polynucleotide of the sequence of the antibody of one or more SSEA3/SSEA4/GloboH. These compositions can additionally comprise well known appropriate carrier in technique, such as pharmaceutically acceptable excipient (including Buffer).

Separated antibody and polynucleotide are also provided.In certain embodiments, separated antibody and polynucleotide are It is substantially pure.

In one embodiment, anti-SSEA3/SSEA4/GloboH antibody is monoclonal antibody.In another embodiment, it mentions For segment (such as Fab, Fab'-SH and F (ab') of anti-SSEA3/SSEA4/GloboH antibody₂Segment).These antibody fragments can It is generated, or can be generated by recombinant technique by the traditional approach of such as enzymatic digestion.These antibody fragments can for chimeric antibody, Humanized antibodies or human antibodies.These segments can be used for reaching diagnosis and therapeutic purposes described below.

Pharmaceutical formulation

Therapeutic composite comprising medical agent of the present invention pass through will have the antibody of wanted purity with it is optionally existing Physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences the 16th Version, Osol, A. compile (1980)) mixing, the preparation in the form of aqueous solution, freeze-dried type or other drying type composites.It is acceptable Carrier, excipient or stabilizer are non-toxic to recipient under used dosage and concentration, and including buffer, such as phosphoric acid Salt, citrate, histidine and other organic acids；Antioxidant, including ascorbic acid and methionine；Preservative (such as ten Eight alkyl dimethyl benzyl ammonium chlorides, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzene first Alcohol, P-hydroxybenzoic acid alkyl ester (such as methyl p-hydroxybenzoate or propylparaben), catechol, resorcinol, Cyclohexanol, 3- amylalcohol and metacresol)；Low molecular weight (being less than about 10 residues) polypeptide；It is protein, such as seralbumin, bright Glue or immunoglobulin；Hydrophilic polymer, such as polyvinyl pyrrolidone；Amino acid, such as glycine, bran amic acid, day Winter amic acid, histidine, spermine acid or from amino acid；Monosaccharide and disaccharide and other sugar, including glucose, mannose or dextrin；Chelating Agent, such as EDTA；Sugar, such as sucrose, mannitol, trehalose or D-sorbite；Salt-forming counterion, such as sodium；Metal is wrong It closes object (such as zinc-albumen misfit object)；And/or non-ionic surfactant, such as TWEEN^TM、PLURONICS^TMOr poly- second two Alcohol (PEG).

Composite herein can also contain have more than it is a kind of for treatment specific adaptations disease needed for reactive compound, including (but being not limited to) has the active they's object of supplement having no adverse effect each other.These molecules are suitble to can effectively achieve expection The amount of purpose, which combines, to be existed.

For example, active constituent can be also trapped in microcapsules, such as by condensation technique or pass through interfacial polymerization institute The microcapsules of preparation, such as respectively hydroxy-methyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules； Colloidal drug delivery system (such as liposome, albumi microspheres, microemulsion, nano particle and Nano capsule) or huge lotion In.These technologies are disclosed in Remington's Pharmaceutical Sciences the 16th edition, and Osol, A. are compiled in (1980).

The composite being intended in vivo offeing medicine must be sterile.It can be easy to realize by aseptic filter membrane filtering.

Extended release preparation can be prepared.The appropriate example of extended release preparation includes consolidating containing immunoglobulin of the present invention The semipermeability matrix of body hydrophobic polymer, these matrix are the form of formed article, such as film or microcapsules.Persistently release The example for putting type matrix includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylate) or poly- (vinyl alcohol)), gathers Copolymer, the nondegradation of lactic acid lactide (U.S. Patent No. 3,773,919), L- Vetsin and γ ethyl-L- Vetsin ester Ethane-acetic acid ethyenyl ester, degradability lactic acid-ethanol copolymer (such as LUPRON DEPOT^TMIt (is copolymerized by lactic acid-ethanol The Injectable microspheres body of object and Leuprorelin acetate composition)) and poly- D- (-) -3-hydroxybutyrate.Although polymer (such as second Alkene-vinyl acetate and lactic acid-ethanol) so that molecule is discharged 100 days, but the period of certain hydrogel release proteins compared with It is short.When be encapsulated antibody for a long time remain in internal when, may be denaturalized or assemble because being exposed to 37 DEG C of moisture, lead to biology Loss of activity and immunogenicity may be made to change.Depending on the mechanism involved in, rational strategy can be designed to reach stabilization Change.For example, if discovery aggregation of multiple is to exchange to form intermolecular S -- S via thio-disulfide, modification can be passed through Sulfydryl residue is lyophilized from acid solution, control moisture content, uses suitable additives and exploitation particular polymers substrate combination Object reaches stabilization.

Medical composition of the invention can be used for treating, inhibit, postpone progress, prevention/delay recurrence, improve or prevention with The unconventionality expression and/or the related disease of activity of SSEA3/SSEA4/GloboH and SSEA3/SSEA4/GloboH related protein Disease, illness or symptom, including but not limited to cancer, disorder of muscle, ubiquitin path correlated inheritance illness, immune/inflammation illness, Nervous disorders and other ubiquitin path associated diseases.

In an aspect, blocking antibody of the invention has specificity to SSEA3/SSEA4/GloboH.

Medical composition of the invention can use in the treatment individually or with other combination of compositions.For example, this hair Bright antibody can offer medicine altogether with other antibody and/or adjuvant/therapeutic agent (such as steroids).For example, antibody of the present invention exists It can be combined with antiphlogistic and/or preservative in therapeutic scheme, such as treat any disease as described herein, including cancer, muscle Illness, ubiquitin path correlated inheritance illness, immune/inflammation illness, nervous disorders and other ubiquitin path associated diseases.The above institute These combination treatments illustrated include that (two of them or two or more medicaments are included in same or separated composite to combination administration In), and administration is separated, in the case, the administration of antibody of the present invention can carry out before adjuvant therapy administration and/or later.

Medical composition (and auxiliary therapeutical agent) of the invention can pass through any appropriate ways (including parenteral, subcutaneous, abdomen In film, intrapulmonary and intranasal) dispensing, and if desired local treatment is then intralesional dispensing.Parenteral infusion includes intramuscular, quiet In arteries and veins, in intra-arterial, peritonaeum or subcutaneous administration.In addition, medical composition preferably passes through pulsatile infusion administration, especially in antibody agent In the case where amount decline.It can be administered by any appropriate approach (such as injecting, such as intravenous or subcutaneous injection), this is partly Depending on the short-term or chronicity offerd medicine.

The position of the combination target of antibody of the present invention is contemplated that in Antibody preparation and dispensing.It is intracellular when combining target When molecule, certain embodiments of the present invention provides the antibody or its antigen binding fragment that can be introduced into the cell being located in conjunction with target Section.In one embodiment, antibody of the invention can be used as intracellular antibody and be expressed in into the cell.As used herein term " Intracellular antibody " refers to cell inner expression and can bind selectively to the antibody or its antigen-binding portion thereof of target molecule, such as following Described in document: Marasco, Gene Therapy 4:11-15 (1997)；Kontermann,Methods 34:163-170 (2004)；U.S. Patent No. No. 6,004,940 and No. 6,329,173；Patent Application Publication the 2003/0104402nd Number and PCT Publication case the WO2003/077945th.The cell inner expression of intracellular antibody be by antibody needed for encoding or its resist The nucleic acid of former bound fraction (lacks wild-type leader sequence and usually related with the gene of encoding antibody or antigen-binding fragment Secretion signal) it is introduced into target cell to realize.Can be used it is any nucleic acid introduces to intracellular standard method, including (but not It is limited to) micro-injection, ballistic injection, electroporation, calcium phosphate Shen Dian, liposome and with the reverse transcription disease for carrying nucleic acid of interest Poison, adenovirus, adeno-associated virus and vaccina vectors transfection.

Medical composition of the invention is deployed, is administered in a manner of meeting good medicine practice and administration.In this context The factor of consideration includes treated particular condition, is treated specific mammal, the clinical condition of few patients, illness Other factors known to cause, the delivering site of medicament, medication administration method, dispensing time-histories and doctor.Antibody is not necessarily to but optionally It is deployed together with the medicament for being currently used in prevention or the treatment illness with one or more.The effective quantity of these other medicaments, which regards, to be existed Amount, illness or the type for the treatment of of antibody of the present invention in composite and as discussed above depending on other factors.These medicines Agent is usually used with same dose and using dosing way described herein, or with about the 1% of dosage described herein to 99% uses, or with any dosage and by rule of thumb/be clinically determined as any approach appropriate and use.

When prevention or treatment disease, medical composition (exclusive use or its other medicine with such as chemotherapeutant of the present invention When agent is applied in combination) suitable dosage regard disease type, Antibody types, disease severity and the course of disease to be treated, antibody is thrown Whether medicine is preventative or therapeutic purpose, previous therapies, patient clinical history and to the reaction of antibody and the judgement of attending physician Depending on.Antibody is suitable for disposably or through a series for the treatment of administration patients.Depending on disease type and severity, about 1 μ g/kg is extremely 15mg/kg (such as 0.1mg/kg-10mg/kg) antibody can be the initial candidate dosage of administration patient, no matter for example by one or It repeatedly separately offers medicine or passes through continuous infusion.A kind of typical daily dose can be in about 1 μ g/kg to 100mg/kg or more than 100mg/ In the range of kg, this is depending on factor referred to above.For the repetition of a couple of days or longer time dispensing, depending on symptom Fixed usually sustainable treatment, until disease symptoms appearance to be inhibited.A kind of Exemplary dosage of antibody about 0.05mg/kg extremely In the range of about 10mg/kg.It therefore, can be by one or more agent of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg Amount (or any combination thereof) administration patient.These dosage can intermittently administration, such as weekly or three weeks every (such as patient is connect By about two to about 20 times or the antibody of for example, about six times dosage).Initially can administration higher load dosage, then can administration one or Multiple relatively low-dose.Illustrative dosage regimen includes the antibody of administration about 4mg/kg initial loading dose, subsequent administration about 2mg/ The antibody of the maintenance dose weekly of kg.However, other dosage regimens can be used.The process of this therapy be easy to through well-known technique and Research and application.

Product

In another aspect of the invention, provide a kind of containing can be used for treating, prevent and/or diagnose the object of above-mentioned illness The product of matter.The product includes container and the label or package insert for investing or lying in the container.Suitable container packet It includes such as bottle, bottle, syringe.Container can be formed by such as glass or plastic a variety of materials.The container accommodates single Solely composition or the composition with another effectively treatment, prevention and/or the combination of compositions for diagnosing symptom, and the container can With sterile inlet port (such as the container can for intravenous solution bag or with can by hypodermic needle perforate plug it is small Bottle).At least one of composition activating agent is antibody of the invention.Label or package insert indicate that the composition is used for Treat selected symptom.In addition, the product may include the first container that (a) includes composition, wherein the composition includes this The antibody of invention；And the second container of composition (b) is included, wherein the composition includes another cytotoxic agent or treatment Agent.Product in the embodiment of the present invention can additionally comprise package insert, and the package insert indication composition can be used for Treat very pathology.Alternatively or in addition, the product can further include one comprising pharmaceutically acceptable buffer (such as Injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), Lin Geershi solution (Ringer's solution) and glucose are molten Liquid) second (or third) container.Its other substance needed for can also comprising for business and user's viewpoint, including its His buffer, diluent, filter, needle and syringe.

The following are the examples of method and composition of the invention.It will be appreciated that consider general remark provided above, it can be real Apply various other embodiments.

In some embodiments, provided glycan conjugate, immunogenic composition or vaccine are suitable for treatment or examine Disconnected cancer, including but not limited to auditory nerve tumor, gland cancer, adrenal, cancer of anus, angiosarcoma (angiosarcoma) (such as lymphangioendothelial sarcoma, lymphatic endothelia sarcoma, angiosarcoma (hemangiosarcoma)), adnexal carcinoma, benign monoclonal globulin Disease, cancer of bile ducts (such as cholangiocarcinoma), bladder cancer, breast cancer (such as breast, papillocarcinoma of breast tumor, breast cancer, breast medullary substance Cancer), the cancer of the brain (such as meningioma；Glioma, such as astrocytoma, oligodendroglia tumor；Medulloblast Tumor), bronchiolar carcinoma, carcinoid tumor, cervix cancer (such as adenocarcinoma of cervix), choriocarcinoma, chordoma, craniopharyngioma, colorectum Cancer (such as colon and rectum carcinoma, colorectal adenocarcinoma), epithelioma, ependymoma, endotheliosarcoma (such as Kaposi sarcoma (Kaposi's sarcoma), multiple idiopathic hemorrhagic sarcoma), carcinoma of endometrium (such as uterine cancer, sarcoma of uterus), food Road cancer (such as esophageal adenocarcinoma, Ba Leiteshi gland cancer (Barrett's adenocarinoma)), ewing's sarcoma (Ewing Sarcoma), her thermophilic erythremia of cancer eye (such as intraocular melanoma, retinoblastoma), familial, gallbladder cancer, Gastric cancer (such as sdenocarcinoma of stomach), gastrointestinal stromal tumors (GIST), head and neck cancer (such as Head and neck squamous cell carcinoma), carcinoma of mouth (such as mouth Chamber squamous cell carcinoma (OSCC), throat cancer (such as laryngocarcinoma, pharynx cancer, nasopharynx cancer, oropharyngeal cancer)), hemopoietic cancer (such as white blood Disease, such as acute lymphatic leukemia (ALL) (such as B cell ALL, T cell ALL), acute myeloid leukemia (AML) (such as B cell AML, T cell AML), chronic myeloid leukaemia (CML) (such as B cell CML, T cell CML) And chronic lymphocytic leukemia (CLL) (such as B cell CLL, T cell CLL)；Lymthoma, such as Hodgkin lymphoma (Hodgkin lymphoma；HL) (such as B cell HL, T cell HL) and non-Hodgkin lymphoma (NHL) (such as B cell NHL, Such as diffusivity large celllymphoma (DLCL) (such as diffusivity large B cell lymphoid tumor (DLBCL)), follicular lymphoma, slow Property lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), lymphoma mantle cell (MCL), marginal zone B cells Lymthoma (such as mucosa-associated lymphoid tissue (MALT) lymthoma, knot marginal zone B-cell lymphoma, marginal zone B cells lymph Tumor), Primary mediastinal B-cell lymthoma, Burkitt lymphoma (Burkitt lymphoma), lymphoplasmacytic lymphoma (also I.e. " Walden Si Telunshi macroglobulinemia (Macroglobulinemia) "), hairy cell leukemia (HCL), immunoblast large celllymphoma, forerunner B lymphoblastoma lymphoma and primary central nervous system (CNS) Lymthoma；And T cell NHL, such as forerunner T lymphoblastoma lymphoma/leukaemia, Peripheral T-cell Lymphoma (PTCL) (example As skin T cell lymphoma (CTCL) (such as mycosis fungoides, Sai Zhalai syndrome (Sezary syndrome)), blood vessel are exempted from Epidemic disease mother cell t cell lymphoma, the outer natural killer t cell lymphoma of knot, enteropathy-type T cell lymphoma, subcutaneous rouge layer inflammation sample T are thin Born of the same parents' lymthoma, pleomorphism large celllymphoma)；The mixing of one or more above-mentioned leukaemia/lymthomas；And Huppert's disease (MM)), heavy chain disease (such as α chain disease, γ chain disease, μ chain disease), hemangioblastoma, inflammatory myofibroblast Property tumour, immunocyte amyloidosis, cancer kidney (such as the nephroblastoma, also known as Weir nurse Si Shi tumour (Wilms'tumor), clear-cell carcinoma), liver cancer (such as hepatocellular carcinoma (HCC), malignant liver), lung cancer (such as bronchiolar carcinoma, Small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of lung), leiomyosarcoma (LMS), mastocytosis (such as systemic mastocytosis), osteomyelodysplasia syndrome (MDS), celiothelioma, myeloproliferative illness (MPD) (such as true property erythremia (PV), primary thrombocytosis (ET), unexplained bone marrow cell metaplasia (AMM) (also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukaemia (CML), it is chronic it is thermophilic in Property leukaemia (CNL), thermophilic Yihong leukocytosis syndrome (HES)), neuroblastoma, neurofibroma (such as 1 type or 2 Type neurofibroma (NF), schwann's tumor are sick (schwannomatosis)), neuroendocrine cancer is (such as in stomach and intestine pancreas nerve Secreting tumor (GEP-NET), carcinoid tumor), osteosarcoma, oophoroma (such as cystadenocarcinoma, ovary embryonal carcinoma, adenocarcinoma ovaries), nipple Shape gland cancer, cancer of pancreas (such as mamillary mucus anything superfluous or useless (IPMN), islet-cell tumour in cancer of pancreas, pipe), carcinoma of penis (such as sun Stem and the osteitis deformans disease (Paget's disease) of scrotum, pinealoma, intramedullary primitive neuroectodermal tumor (PNT), forefront Gland cancer (such as adenocarcinoma of the prostate), the carcinoma of the rectum, rhabdomyosarcoma, salivary-gland carcinoma, cutaneum carcinoma (such as squamous cell carcinoma (SCC), angle Change acanthoma (acanthomata) (KA), melanoma, basal-cell carcinoma (BCC)), carcinoma of small intestine (such as adnexal carcinoma), soft tissue sarcoma it is (such as pernicious Fibrous histiocytoma (MFH), embryonal-cell lipoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, mucus Sarcoma), sebaceous glands carcinoma, syringocarcinoma, synovialoma, carcinoma of testis (such as seminoma, embryonal carcinoma of testis), thyroid cancer (example Such as thyroid papillary carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid cancer), carcinoma of urethra, carcinoma of vagina and carcinoma of vulva (example Such as the osteitis deformans of vulva).In certain embodiments, provided glycan conjugate, immunogenic composition or vaccine are suitable For treating the cancer of the brain, lung cancer, breast cancer, carcinoma of mouth, cancer of the esophagus, gastric cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, colon cancer, renal cancer, bone Cancer, cutaneum carcinoma, cervix cancer, oophoroma and prostate cancer.

It, can be via suitable approach as described above by a effective amount of this paper institute in order to execute treatment method as described herein State any glycan conjugate or immunogenic composition or vaccine administration individual in need for the treatment of.Individual, such as human individual, It can be with cancer, suspect with cancer or cancer-prone patient.The glycan conjugate or IMMUNOGENIC COMPOSITION of administration individual The amount of object can effectively induce specificity for the immune response of conjugate or the glycan moiety in composition.In some embodiments In, the amount of glycan conjugate or immunogenic composition is enough to induce inhibition growth of cancers and/or reduces the immune anti-of tumor mass It answers.In other embodiments, the amount of glycan conjugate or immunogenic composition can effectively delay the breaking-out or drop of Target cancers It is low the risk of cancer occur.Glycan conjugate, immunogenic composition or vaccine are provided necessary to effective quantity really to reach The amount of cutting will be different because of individual, this is depending on for example following: individual species, age and general status, side effect or illness it is tight Weight degree, the status of specific compound, dispensing mode and similar factor.The delivering of wanted dosage can for three times a day, one day two It is secondary, once a day, every other day, every three days, weekly, every two weeks, every three weeks or every four weeks.In certain embodiments, it Dosage can be used repeatedly dispensing delivering (such as twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, 11 It is secondary, 12 times, ten three times, 14 times or offer medicine more than 14 times).

In certain embodiments, provided glycan conjugate, immunogenic composition or vaccine are thrown to 70kg adult It may include each unit dosage forms about 0.0001mg to about 3000mg, about 0.0001mg to about with effective quantity one or more times 2000mg, about 0.0001mg to about 1000mg, about 0.001mg to about 1000mg, about 0.01mg to about 1000mg, about 0.1mg extremely About 1000mg, about 1mg are to about 1000mg, about 1mg to about 100mg, about 10mg to about 1000mg, or about 100mg to about 1000mg Compound.

In certain embodiments, provided glycan conjugate, immunogenic composition or vaccine can be oral or parenteral, To be enough to deliver about daily per kilogram whose body weight 0.001mg to about 100mg, about 0.01mg to about 50mg, preferably from about 0.1mg extremely About 40mg, preferably from about 0.5mg are to about 30mg, about 0.01mg to about 10mg, about 0.1mg to about 10mg and more preferably about 1mg to about The dosage level of 25mg, one day administration one or more times, to obtain wanted therapeutic effect.

It should be appreciated that dosage range as described herein is provided about to glycan conjugate, immunogene provided by adult administration The guidance of property composition or vaccine.The amount of administration such as child or adolescent can by doctor or those who familiarize themselves with the technology determine and The amount or identical as the amount of administration adult of administration adult can be lower than.

It should also be understood that provided glycan conjugate, immunogenic composition or vaccine can be with one or more other treatments Activating agent combines administration.Provided glycan conjugate, immunogenic composition or vaccine can with improve its biological usability, subtract Less and/or modifies its metabolism, it is inhibited to drain and/or modify the other treatment activating agent of its distribution in vivo combination administration. It should also be understood that therapy used can be reached to be acted on for identical illness, and/or it may achieve different role.

Provided glycan conjugate, immunogenic composition or vaccine can be same with one or more other treatment activating agents When, the rear of its or rear administration.In general, each medicament will according to the dosage according to determined by the medicament and/or time-histories come Administration.Additionally, it is understood that other treatment activating agent used in this combination can in single composition administration or in difference together Administration is separated in composition.Specific combination used in therapy will consider that the compounds of this invention is compatible with other treatment activating agent Property and/or the wanted therapeutic effect to be reached.In general, it is contemplated that the dosage of other treatment activating agent used in combination does not surpass Cross amount when its individual use.In some embodiments, combination dosage will be less than individual usage amounts.

In certain embodiments, provided glycan conjugate, immunogenic composition or vaccine and one or more this paper Other described medical agents combine administration.In certain embodiments, other medical agents are anticancer agent.It is anti-that anticancer agent covers biological therapy Cancer agent and chemotherapeutant.

Exemplary biological treatment anticancer agent includes but is not limited to that interferon, cytohormone are (such as tumor necrosis factor, dry Disturb plain α, interferon gamma), vaccine, hemopoieticgrowth factor, single plant serum therapy, immunostimulant and/or immunomodulator (such as IL-1, IL-2, IL-4, IL-6 or IL-12), immune cell growth factor (such as GM-CSF) and antibody (such as Trastuzumab (Herceptin) (Herceptin (trastuzumab)), T-DM1, Arastin (AVASTIN) (bevacizumab (bevacizumab)), Chinese mugwort must appropriate (ERBITUX) (Cetuximab (cetuximab)), dimension gram for than (Vectibix) (Pa Ni Monoclonal antibody (panitumumab)), Mabthera (Rituxan) (rituximab), husky (the Bexxar) (tositumomab of hectogram (tositumomab)))。

Exemplary chemical therapeutic agent includes but is not limited to antiestrogenic (such as tamoxifen (tamoxifen), Reynolds former times Phenol (raloxifene) and megestrol acetate (megestrol)), LHRH agonist (such as cream Si Kelin (goscrclin) and bright third Vertical moral (leuprolide)), antiandrogen (such as Flutamide (flutamide) and Bicalutamide (bicalutamide)), light Motivation therapy (such as not appropriate amber fragrant (vertoporfin) (BPD-MA), phthalocyanine (phthalocyanine), emulsion Pc4 and De-methoxy-hypocrellin A (demethoxy-hypocrellin A) (2BA-2-DMHA)), mustargen (nitrogen Mustards) (such as cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), Trofosfamide (trofosfamide), Chlorambucil (chlorambucil), estramustine (estramustine) and melphalan (melphalan)), nitroso ureas (nitrosoureas) (such as Carmustine (carmustine) (BCNU) and lomustine (lomustine) (CCNU)), alkyl sulfonic ester (such as busulfan (busulfan) and Treosulfan (treosulfan)), three Nitrence (such as Dakar bar piperazine (dacarbazine), Temozolomide (temozolomide)), compound containing platinum (such as cis-platinum (cisplatin), carboplatin (carboplatin), oxaliplatin (oxaliplatin)), (such as Changchun is new for vinca alkaloids Alkali (vincristine), vincaleukoblastinum (vinblastine), eldisine (vindesine) and Vinorelbine (vinorelbine)), taxoid (such as Paclitaxel or Paclitaxel equivalent, the white egg of such as nanoparticle White combination Paclitaxel (A Bula is raw (Abraxane)), (DHA- is too for docosahexaenoic acid combination Paclitaxel Flat ocean taxol, Ta Kepuxin (Taxoprexin)), poly- Vetsin salt combination Paclitaxel (PG- Paclitaxel, The poly- Vetsin of Paclitaxel, CT-2103, XYOTAX), tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 combine To three Paclitaxel molecules), Paclitaxel-EC-1 (Paclitaxel be bound to identification erbB2 peptide EC- 1) and the Paclitaxel that combines of glucose, such as 2'- Paclitaxel 2- grape piperazine is muttered glycosyl succinic acid methyl esters；It is more Xi Tasai (docetaxel), taxol (taxol)), table podophyllin (epipodophyllins) (such as Etoposide (etoposide), etoposide phosphate (etoposide phosphate), Teniposide (teniposide), topotecan (topotecan), 9-aminocamptothecin, open it is general for health (camptoirinotecan), it is Irinotecan (irinotecan), gram vertical That support (crisnatol), mitomycin C (mytomycin C)), antimetabolite, DHFR inhibitor (such as methotrexate (methotrexate), dichloro talk endlessly acyl Vetsin (dichloromethotrexate), Trimetrexate (trimetrexate), according to Up to Qu Sha (edatrexate)), IMP dehydrogenation enzyme inhibitor (such as mycophenolic acid (mycophenolic acid), Tiazofurine (tiazofurin), virazole (ribavirin) and EICAR), ribonucleotide reductase inhibitors (such as hydroxyurea (hydroxyurea) and Deferoxamine (deferoxamine)), uracil analogues (such as 5 FU 5 fluorouracil (5-FU), floxuridine (floxuridine), deoxyfluorouridine (doxifluridine), thunder replace Qu Sai (ratitrexed), tegafur-uracil (tegafur-uracil), capecitabine (capecitabine)), analogue of cytosine (such as cytarabine (cytarabine) (ara C), cytarabin (cytosine arabinoside) and fludarabine (fludarabine)), purine analogue (such as purinethol (mercaptopurine) and thioguanine (Thioguanine)), vitamin D 3 analogs (such as EB 1089, CB 1093 and KH 1060), prenylation inhibitor (example Such as Lovastatin (lovastatin)), dopamine stimulated conductivity neurotoxin (such as 1- methyl 4-phenyl pyrrole ingot ion), cell Cycle inhibitor (such as staurosporin (staurosporine)), D actinomycin D (actinomycin) (such as D actinomycin D D, D actinomycin D d), bleomycin (bleomycin) (such as Bleornycin A2, Bleomycin B2, Peplomycin (peplomycin)), anthracycline (anthracycline) (such as daunomycin (daunorubicin), adriamycin (doxorubicin), pegylated liposomal adriamycin, angstrom reach mycin (idarubicin), epirubicin (epirubicin), pirarubicin (pirarubicin), a left side are soft than star (zorubicin), mitoxantrone (mitoxantrone)), MDR inhibitor (such as Verapamil (verapamil)), Ca²⁺Atpase inhibitor (such as malicious Hu trailing plants Bu Su (thapsigargin)), Imatinib (imatinib), Distaval (thalidomide), lenalidomide (lenalidomide), tyros kinase inhibitor (such as Axitinib (axitinib) (AG013736), Bosutinib (bosutinib) (SKI-606), Si Dinibu (cediranib) (RECENTINTM, AZD2171), Dasatinib (dasatinib)(BMS-354825), Erlotinib (erlotinib)Ji Fei For Buddhist nun (gefitinib)Imatinib (imatinib) (CGP57148B, STI-571), Lapatinib (lapatinib)Lestaurtinib (lestaurtinib) (CEP- 701), linatinib (neratinib) (HKI-272), nilotinib (nilotinib)Smasani (semaxanib) (Si Maxini (semaxinib), SU5416), Sutent (sunitinib) ( SU11248), appropriate Sai Lanni (toceranib)Vande Thani (vandetanib) (ZD6474), all tower indigo plant Buddhist nuns (vatalanib) (PTK787, PTK/ZK), Herceptin (trastuzumab)Bevacizumab (bevacizumab)Rituximab (rituximab)Cetuximab (cetuximab)Victibix (panitumumab)Ranibizumab (ranibizumab)Nilotinib (nilotinib)Sorafenib (sorafenib)Everolimus (everolimus)Alemtuzumab (alemtuzumab)WAY-CMA 676 Austria azoles Meter Xing (gemtuzumab ozogamicin)Tamiros (temsirolimus)The more Weis of ENMD-2076, PCI-32765, AC220, lactic acid replace Buddhist nun (dovitinib lactate) (TKI258, CHIR-258), BIBW 2992 (TOVOKTM), SGX523, PF-04217903, PF-02341066, PF- 299804、BMS-777607、ABT-869、MP470、BIBF 1120AP24534、JNJ- 26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, for Wo Zhani (tivozanib) (AV-951), OSI-930, MM-121, XL-184, XL-647 and/or XL228), proteasome inhibitor (such as bortezomib (bortezomib) (Bortezomib (Velcade))), mTOR inhibitors (such as rapamycin (rapamycin), tamiros (temsirolimus) (CCI-779), everolimus (everolimus) (RAD-001), AP 23573 (ridaforolimus)、AP23573(Ariad)、AZD8055(AstraZeneca)、BEZ235(Novartis)、BGT226 (Norvartis)、XL765(Sanofi Aventis)、PF-4691502(Pfizer)、GDC0980(Genetech)、SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen (oblimersen), gemcitabine (gemcitabine), carminomycin (carminomycin), formyl tetrahydrofolic acid (leucovorin), pemetrexed (pemetrexed), cyclophosphamide (cyclophosphamide), Dakar bar piperazine (dacarbazine), procarbazine (procarbizine), prednisolone (prednisolone), dexamethasone (dexamethasone), Kapp heat new (campathecin), plicamycin (plicamycin), asparaginase (asparaginase), aminopterin-induced syndrome (aminopterin), methotrexate (methopterin), porfiromycin (porfiromycin), melphalan (melphalan), leurosidine (leurosidine), leurosine (leurosine), Chlorambucil (chlorambucil), tributidine (trabectedin), procarbazine (procarbazine), Di Sidemo come (discodermolide), carminomycin (carminomycin), aminopterin-induced syndrome and hexamethyl melamine (hexamethyl melamine).

In certain embodiments, individual treated is mammal.In certain embodiments, individual is the mankind.At certain In a little embodiments, individual is performing animal, such as dog, cat, ox, pig, horse, sheep or goat.In certain embodiments, individual is Companion animals, such as dog or cat.In certain embodiments, individual is livestock animals, such as ox, pig, horse, sheep or goat.? In some embodiments, individual is zoo animal.In another embodiment, individual is research animal, such as rodent, dog Or non-human primate.In certain embodiments, individual is that non-human turns to grow genetic animal, such as turn to grow DNA murine or Turn to grow gene pig.

Example

Including following instance to show the preferred embodiment of the present invention.Skilled artisan will understand that in following instance Revealed technology represents the inventors discovered that playing the technology of good action in the present invention is implemented, and therefore can be considered and constitute it Preferred embodiment.However, according to the present invention, skilled artisan will understand that, without departing substantially from spirit and scope of the invention In the case where can to revealed specific embodiment, many modifications may be made and still obtains same or like result.

The illustrative synthesis of example 1:SSEA3 analog

The chemical enzyme' s catalysis of A:SSEA3 analog-amino

Process 1. synthesizes SSEA3 analog-amino by regenerative system

Merge compound Gb4 analog, ATP, UTP, gala sugar analogue, phosphoenolpyruvate ester, magnesium chloride and enzyme Galactokinase (GalK), UDP sugar pyrophosphorylase (AtUSP), β -1,3- galactosyltransferase (LgtD), pyruvate kinase (PK) and inorganic pyrophosphatase (PPA) is in solution, starts to react at room temperature, and controlling pH value is 7.0, and passes through film layer Analysis plate monitoring reaction is until no longer observe product.After the reaction was completed, it is removed in reaction mixture by heating 30 minutes Protein is then centrifuged and is filtered with 0.22 μM of filter.Then pass through C-18 gel chromatography filtrate.Collect it is molten from part and It is monitored by membrane chromatographic plate.

The illustrative synthesis of example 2:SSEA4 analog

The chemical synthesis of A:SSEA4-Gc- amino

Process 2: SSEA4-Gc- amino is synthesized by chemical synthesis

By powdered molecular sieve (4A, 0.5g) be added to receptor 3 (93mg, 0.045mmol) and imide ester 6 (76mg, 0.068mmol) in 6mL methylene chloride (CH₂Cl₂) in solution in.Mixture is stirred at room temperature 2 hours.Be then cooled to- After 10 DEG C, add TMSOTf (5 μ L, 0.03mmol), and mixture is stirred overnight 5 DEG C (cold houses).Reaction mixture passes through Addition triethylamine (0.5mL) is quenched, and uses CH₂Cl₂It dilutes and is filtered via Celite pad.Filtrate saturated sodium bicarbonate (NaHCO₃) aqueous solution washing, through sodium sulphate (Na₂SO₄) water removal, filtering and concentration.(liquid is purged with by silica gel chromatography For 50-100% ethyl acetate/n-hexane) purifying residue, six sugar 7 are obtained, are contaminated with from the miscellaneous of disaccharides imide ester 6 Matter.Yield (90mg, 68%) is measured by NMR.

Zinc powder (1g) is added to six sugar 7 (90mg, 0.03mmol) in the solution in glacial acetic acid (5.0mL) and stirring is mixed It closes object 1 to 2 hour, according to membrane chromatographic plate analysis until compound 7 exhausts.Reaction mixture is diluted with methylene chloride, via Celite pad is filtered and is concentrated under reduced pressure.Residue is dissolved in pyridine/acetic anhydride (1:1,2.0mL), stirring 1 hour and Concentration.Residue is purified by silica gel chromatography.To be acylated substance be dissolved in anhydrous methylene chloride and methanol (2: 8,10mL) it is handled in and with sodium methoxide (45mg).It is stirred at room temperature after 4 hours, adds water (0.2mL), and stir gained Mixture 16 hours.Reaction mixture is neutralized with cation exchange resin amberlyst IR-120, filtering and concentration.By inverse Phase chromatography (RP-18) purifies residue.

By palladium dydroxide (20%, in charcoal, 50mg) and purified be added to methanol/water/acetic acid (10:10:0.5, 6mL) in mixed liquor, and at the stirring under hydrogen of one atmospheric pressure of room temperature 16 hours.Mixture is filtered and is concentrated via Celite pad. Residue is purified by reverse-phase chromatography method, obtains compound 8 (17mg, 43%).

The chemical enzyme' s catalysis of B:SSEA4 analog-amino

Process 3. generates SSEA4 analog-amino synthesis path by regenerative system

SSEA4 analog-amino is synthesized via the enzymatic regeneration strategy as described in process 3.In such a system, make N- Acetyl-D-MANNOSE amine derivative and pyruvate react and to be converted to n acetylneuraminic acid n by aldolase catalysis similar Object then merges in regenerative system with Gb5- amino (J.Am.Chem.Soc.2013,135,14831-14839), obtains model SSEA4 analog-amino of example.

Reaction condition is described in detail as follows: Gb5- amino (18 μm of ol), CTP (5 μm of ol), N- acetyl-D-MANNOSE amine spread out Biological (27 μm of ol), Sodium Pyruvate (81 μm of ol), PEP (55 μm of ol) and ATP (5 μm of ol) are dissolved in 50mM Tris-HCl buffering In liquid (pH 8.0).By α-(2,3)-sialyltransferase (20 units), acetylneuraminate aldolase (20 units), CMK enzyme (10 A unit), Pykf enzyme (10 units), PPA enzyme (10 units) and Pmcss enzyme (10 units) be added in solution, and 37 DEG C are reacted 8 hours and are monitored by membrane chromatographic plate.At the end of reaction, become enzyme by heating 5 minutes at 100 DEG C Property.Desired SSEA4 analog-amino is column purified by G25, DEAE and SP, yield about 80%.

SSEA4 analog-amino¹H NMR

B-1.SSEA4- amylamine (R_N=NHAc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.94 (d, J=3.8Hz, 1H), 4.72 (d, J=8.5Hz, 1H), 4.54-4.50 (m, 3H), 4.40 (t, J=6.4Hz, 1H), 4.27 (d, J=2.0Hz, 1H), 4.20 (d, J=2.8Hz, 1H), 4.10-3.54 (m, 37H), 3.34-3.31 (m, 1H), 3.02 (t, J=7.6Hz, 2H), 2.78 (dd, J=12.4,4.6Hz, 1H), 2.05 (m, 6H),1.80(t,12.2Hz,1H),1.74-1.67(m,4H),1.51-1.45(m,2H)

B-2.Neu5Gc_SSEA4- amylamine (R_N=NHGc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.89 (d, J=3.6Hz, 1H), 4.66 (d, J=8.2Hz, 1H), 4.52-4.45 (m, 3H), 4.37 (t, J=6.8Hz, 1H), 4.23 (d, J=3.2Hz, 1H), 4.15 (d, J=2.8Hz, 1H), 4.10-3.48 (m, 35H), 3.27 (m, 1H), 2.98 (t, J=7.6Hz, 2H), 2.73 (dd, J=4.8,12.4Hz, 1H), 2.00 (s, 3H), 1.77 (t, J=12.0Hz, 1H), 1.72-1.61 (m, 4H), 1.48-1.39 (m, 2H).

B-3.Ac- alkynyl _ SSEA4- amylamine (R_N=NHCOC₂H₄C≡CH,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.89 (d, J=4.0Hz, 1H), 4.67 (d, J=8.4Hz, 1H), 4.52-4.45 (m, 3H), 4.37 (t, J=6.4Hz, 1H), 4.23 (d, J=2.4Hz, 1H), 4.08-3.54 (m, 38H), 3.28 (m, 1H), 2.99 (t, J=7.6Hz, 2H), 2.53-2.4 (m, 4H), 2.37 (s, 1H), 2.01 (s, 3H), 1.77 (t, J=12.0Hz, 1H),1.72-1.62(m,4H),1.49-1.41(m,2H)。

B-4.Ac- fluoride _ SSEA4- amylamine (R_N=NHCOCH₂F,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.90 (d, J=46.4Hz, 2H), 4.90 (d, J=4.0Hz, 1H), 4.67 (d, J= 8.8Hz, 1H), 4.53-4.46 (m, 3H), 4.37 (t, J=6.8Hz, 1H), 4.24 (d, J=2.8Hz, 2H), 4.16 (d, J= 3.2Hz, 1H), 4.09-3.51 (m, 34H), 3.28 (m, 1H), 2.99 (t, J=7.2Hz, 1H), 2.75 (dd, J=4.8, 12.4Hz, 1H), 2.01 (s, 3H), 1.79 (t, J=12.0Hz, 1H), 1.72-1.62 (m, 4H), 1.48-1.40 (m, 2H).

B-5.Ac- phenyl _ SSEA4- amylamine (R_N=NHCOCH₂Ph,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.39-7.30 (m, 5H), 4.90 (d, J=4.0Hz, 1H), 4.66 (d, J= 8.4Hz, 1H), 4.52-4.46 (m, 3H), 4.37 (t, J=6.8Hz, 1H), 4.23 (d, J=2.8Hz, 1H), 4.15 (d, J= 3.2Hz, 1H), 4.08-3.47 (m, 38H), 3.36 (dd, J=1.6,9.2Hz, 1H), 3.28 (m, 1H), 2.99 (t, J= 7.6Hz, 2H), 2.73 (dd, J=4.8,12.4Hz, 1H), 2.00 (s, 3H), 1.76 (t, J=12.0Hz, 1H), 1.72-1.61 (m,4H),1.51-1.40(m,2H)。

B-6.Ac- repeatedly nitrogen base _ SSEA4- amylamine (R_N=NHCOCH₂N₃,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.88 (d, J=3.6Hz, 1H), 4.66 (d, J=8.4Hz, 1H), 4.52-4.44 (m, 3H), 4.36 (t, J=6.4Hz, 1H), 4.23 (d, J=2.4Hz, 1H), 4.08-3.54 (m, 35H), 3.27 (m, 1H), 2.98 (t, J=7.2Hz, 2H), 2.73 (dd, J=4.8,12.4Hz, 1H), 2.00 (s, 3H), 1.77 (t, J=12.4Hz, 1H),1.72-1.60(m,4H),1.48-1.39(m,2H)。

B-7.5'- repeatedly nitrogen base _ SSEA4- amylamine (R_N=N₃,R₁₀=OH)

¹H NMR(400MHz,D₂O): δ 4.90 (d, J=3.6Hz, 1H), 4.67 (d, J=8.4Hz, 1H), 4.51-4.47 (m, 3H), 4.37 (t, J=6.4Hz, 1H), 4.23 (d, J=2.8Hz, 1H), 4.15 (d, J=3.2Hz, 1H), 4.08-3.44 (m, 35H), 3.31-3.27 (m, 1H), 2.99 (t, J=7.2Hz, 1H), 2.73 (dd, J=4.8,12.4Hz, 1H), 2.01 (s, 3H), 1.76 (t, J=12.0Hz, 1H), 1.72-1.63 (3,4H), 1.48-1.41 (m, 2H)；HRMS(ESI-TOF,M-H-) C₄₆H₇₈N₅O₃₃Calculated value 1228.4579, experiment value 1228.4621.

B-8.9'- repeatedly nitrogen base _ SSEA4- amylamine (R_N=NHAc, R₁₀=N₃)

¹H NMR(400MHz,D₂O) δ 4.85 (d, J=3.8Hz, 1H), 4.67 (d, J=8.4Hz, 1H), 4.51-4.44 (m, 3H), 4.37 (t, J=6.4Hz, 1H), 4.23 (d, J=2.8Hz, 1H), 4.10-3.40 (m, 33H), 3.27 (m, 1H), 2.98 (t, J=7.6Hz, 2H), 2.72 (dd, J=4.8,12.8Hz, 1H), 2.00 (s, 3H), 2.00 (s, 3H), 1.75 (t, J =12.4Hz, 1H), 1.72-1.60 (m, 4H), 1.58-1.38 (m, 2H).

B-9.NHBz_SSEA4- amylamine (R_N=NHBz, R₁₀=OH)

¹H NMR(400MHz,D₂O)δ7.80-7.73(m,2H),7.63(m,1H),7.56-7.51(m,2H),4.92(d,J =4.0Hz, 1H), 4.70 (d, J=8.4Hz, 1H), 4.58-4.47 (m, 3H), 4.40 (t, J=6.4Hz, 1H), 4.26 (d, J =2.8Hz, 1H), 4.19 (d, J=3.2Hz, 1H), 4.15-3.53 (m, 36H), 3.31 (m, 1H), 3.01 (t, J=7.6Hz, 2H), 2.82 (dd, J=4.4,12.4Hz, 1H), 2.00 (s, 3H), 1.87 (t, J=12.0Hz, 1H), 1.72-1.60 (m, 4H),1.48-1.39(m,2H)。

C:SSEA4 analog-mercaptan cross-linking reaction

Process 4: SSEA4 analog-mercaptan is synthesized via DTSSP cross-linking reaction

In certain embodiments, DTSSP (2.0eq) and SSEA4 analog-amino (1.0eq) are mixed in 0.1M phosphoric acid In salt buffer pH 7.4 (about 3mg/mL).One night of solution is stirred at room temperature.Then by reaction mixture be warming up to 40 DEG C and It adds DTT (9.0eq).After 40 DEG C are stirred 1.5 hours, it is concentrated in vacuo reaction mixture, and column purified residual by LH-20 Excess obtains white solid SSEA4 analog-mercaptan.(process 4)

SSEA4 analog-mercaptan¹H NMR

C-1:SSEA4- mercaptan (R_N=NHAc, R₁₀=OH)

¹H NMR(400Hz,D₂O) δ 4.88 (d, J=4.0Hz, 1H), 4.65 (d, J=8.5Hz, 1H), 4.50-4.44 (m, 3H), 4.36 (t, J=6.5Hz, 1H), 4.22 (d, J=2.9Hz, 1H), 4.14 (d, J=3.1Hz, 1H), 4.04-3.55 (m, 35H), 3.26 (t, J=8.5Hz, 1H), 3.18 (t, J=6.8Hz, 2H), 2.74-2.70 (m, 3H), 2.49 (t, J=6.8Hz, 2H), 1.994 (s, 3H), 1.992 (s, 3H), 1.75 (t, J=12.2Hz, 1H), 1.61 (tt, J=6.7,6.7HZ, 2H), 1.52 (tt, J=7.1,7.1Hz, 2H), 1.40-1.36 (m, 2H)；

C-2:Neu5Gc_SSEA4- mercaptan (R_N=NHGc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.89 (d, J=3.9Hz, 1H), 4.66 (d, J=8.5Hz, 1H), 4.53-4.43 (m, 3H), 4.36 (t, J=6.5Hz, 1H), 4.22 (d, J=3.0Hz, 1H), 4.15 (d, J=3.1Hz, 1H), 4.11-3.48 (m, 38H), 3.27 (t, J=8.4Hz, 1H), 3.19 (t, J=6.7Hz, 2H), 2.78-2.71 (m, 3H), 2.51 (t, J= 6.7Hz, 2H), 2.00 (s, 3H), 1.78 (t, J=12.1Hz, 1H), 1.61 (q, J=7.1Hz, 2H), 1.52 (q, J= 7.1Hz, 2H), 1.39 (q, J=8.0Hz, 2H).

C-3:Ac- alkynyl _ SSEA4- mercaptan (R_N=NHCOC₂H₄C≡CH,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.94 (d, J=3.9Hz, 1H), 4.72 (d, J=8.4Hz, 1H), 4.58-4.48 (m, 3H), 4.41 (t, J=6.5Hz, 1H), 4.30-4.26 (m, 1H), 4.21 (d, J=3.1Hz, 1H), 4.14-3.54 (m, 37H), 3.32 (t, J=8.6Hz, 1H), 3.24 (t, J=6.8Hz, 2H), 2.83-2.74 (m, 3H), 2.59-2.49 (m, 5H), 2.43 (s, 1H), 2.06 (s, 3H), 1.82 (t, J=12.1Hz, 1H), 1.67 (p, J=6.9Hz, 2H), 1.58 (p, J= 6.9Hz,2H),1.48-1.38(m,2H)。

C-4:Ac- fluoride _ SSEA4- mercaptan (R_N=NHCOCH₂F, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.90 (d, J=46.4Hz, 2H), 4.95 (d, J=4.0Hz, 1H), 4.72 (d, J= 8.5Hz, 1H), 4.59-4.48 (m, 3H), 4.41 (t, J=6.6Hz, 1H), 4.31-4.26 (m, 1H), 4.23-4.18 (m, 1H), 4.14-3.54 (m, 36H), 3.36-3.29 (m, 1H), 3.25 (t, J=6.8Hz, 2H), 2.80 (m, 3H), 2.57 (t, J =6.7Hz, 2H), 2.06 (s, 3H), 1.84 (t, J=12.2Hz, 1H), 1.67 (p, J=6.9Hz, 2H), 1.58 (p, J= 7.0Hz, 2H), 1.43 (q, J=8.3Hz, 2H).

C-5:Ac- phenyl _ SSEA4- mercaptan (R_N=NHCOCH₂Ph, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.48-7.32 (m, 5H), 4.94 (d, J=3.6Hz, 1H), 4.73-4.68 (d, J= 8.4Hz, 1H), 4.52 (m, 3H), 4.41 (t, J=6.4Hz, 1H), 4.29-4.26 (m, 1H), 4.20 (d, J=3.0Hz, 1H), 4.13-3.51 (m, 37H), 3.39 (dd, J=9.0,1.8Hz, 1H), 3.32 (t, J=8.6Hz, 1H), 3.25 (t, J= 6.7Hz, 2H), 2.83-2.74 (m, 3H), 2.56 (t, J=6.7Hz, 2H), 2.04 (s, 3H), 1.80 (t, J=12.1Hz, 1H), 1.67 (q, J=7.2Hz, 2H), 1.57 (q, J=7.1Hz, 2H), 1.48-1.38 (m, 2H).

C-6:Ac- repeatedly nitrogen base _ SSEA4- mercaptan (R_N=NHCOCH₂N₃, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 4.88 (d, J=3.9Hz, 1H), 4.66 (d, J=8.5Hz, 1H), 4.52-4.43 (m, 3H), 4.36 (t, J=6.5Hz, 1H), 4.22 (d, J=3.1Hz, 1H), 4.14 (d, J=3.1Hz, 1H), 4.08-3.47 (m, 38H), 3.26 (t, J=8.4Hz, 1H), 3.19 (t, J=6.8Hz, 2H), 2.74 (m, 3H), 2.51 (t, J=6.7Hz, 2H), 2.00 (s, 3H), 1.76 (t, J=12.1Hz, 1H), 1.61 (q, J=7.1Hz, 2H), 1.53 (p, J=7.0Hz, 2H), 1.38 (q, J=8.3Hz, 2H).

C-7:5'- repeatedly nitrogen base _ SSEA4- mercaptan (R_N=N₃, R₁₀=OH)

¹H NMR(400Hz,D₂O) δ 4.90 (d, J=4.0Hz, 1H), 4.67 (d, J=8.4Hz, 1H), 4.51-4.46 (m, 3H), 4.37 (t, J=6.4Hz, 1H), 4.24 (d, J=2.8Hz, 1H), 4.15 (d, J=2.8Hz, 1H), 4.01-3.44 (m, 35H), 3.28 (t, J=8.4Hz, 1H), 3.21 (t, J=6.8Hz, 2H), 2.78-2.72 (m, 3H), 2.52 (t, J=7.2Hz, 2H), 2.02 (s, 3H), 1.77 (t, J=12.0Hz, 1H), 1.67-1.60 (m, 2H), 1.58-1.50 (m, 2H), 1.43-1.37 (m,2H)

C-8:9'- repeatedly nitrogen base _ SSEA4- mercaptan (R_N=NHAc, R₁₀=N₃)

¹H NMR(400MHz,D₂O) δ 4.90 (d, J=3.9Hz, 1H), 4.68 (d, J=8.5Hz, 1H), 4.48 (dd, J= 13.2,7.9Hz, 3H), 4.37 (t, J=6.5Hz, 1H), 4.26-4.22 (m, 1H), 4.16 (d, J=3.3Hz, 1H), 4.09- 3.44 (m, 36H), 3.31-3.24 (m, 1H), 3.20 (t, J=6.8Hz, 2H), 2.79-2.70 (m, 3H), 2.52 (t, J= 6.7Hz, 2H), 2.02 (d, J=2.0Hz, 6H), 1.76 (t, J=12.1Hz, 1H), 1.63 (p, J=6.9Hz, 2H), 1.54 (p, J=6.9Hz, 2H), 1.39 (q, J=8.3Hz, 2H).

C-9:NHBz_SSEA4- mercaptan (R_N=NHBz, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.80-7.73 (m, 2H), 7.66-7.58 (m, 1H), 7.52 (dd, J=8.4, 7.0Hz, 2H), 4.91 (d, J=3.9Hz, 1H), 4.69 (d, J=8.5Hz, 1H), 4.57-4.44 (m, 3H), 4.38 (t, J= 6.5Hz, 1H), 4.27-4.22 (m, 1H), 4.18 (d, J=3.1Hz, 1H), 4.16-3.52 (m, 36H), 3.29 (t, J= 8.5Hz, 1H), 3.20 (t, J=6.8Hz, 2H), 2.84-2.72 (m, 3H), 2.52 (t, J=6.7Hz, 2H), 2.03 (s, 3H), 1.89 (t, J=12.2Hz, 1H), 1.63 (p, J=6.8Hz, 2H), 1.53 (q, J=7.1Hz, 2H), 1.40 (q, J=8.2Hz, 2H)。

The chemical enzyme' s catalysis of D:SSEA4 analog-allyl

Process 5. generates SSEA4 analog-allyl synthesis path by regenerative system

SSEA4 analog-allyl is synthesized via the enzymatic regeneration strategy as described in process 5.In such a system, make N- acetyl-D-amino mannose derivative reacts with pyruvate and is converted to n acetylneuraminic acid n class by aldolase catalysis Like object, then merges (J.Am.Chem.Soc.2013,135,14831-14839) in regenerative system with Gb5- allyl, obtain Obtain SSEA4 analog-allyl of example.(process 5)

Reaction condition is described in detail as follows: Gb5- allyl (18 μm of ol), CTP (5 μm of ol), N- acetyl-D-amino is sweet Dew sugar derivatives (27 μm of ol), Sodium Pyruvate (81 μm of ol), PEP (55 μm of ol) and ATP (5 μm of ol) are dissolved in 50mM Tris- In HCl buffer (pH 8.0).By α-(2,3)-sialyltransferase (20 units), acetylneuraminate aldolase (20 units), CMK enzyme (10 units), Pykf enzyme (10 units), PPA enzyme (10 units) and Pmcss enzyme (10 units) are added to solution In, and react 8 hours at 37 DEG C and monitored by membrane chromatographic plate.At the end of reaction, by 100 DEG C heat 5 minutes come Make enzyme denaturation.Wanted SSEA4 analog-allyl is column purified by G25, DEAE and SP, yield about 80%.

SSEA4 analog-allyl¹H NMR

D-1.SSEA4- allyl (R₁=OH, R_N=NHAc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 6.00 (m, 1H), 5.40-5.37 (d, J=17.3Hz, 1H), 5.30-5.28 (d, J =10.4Hz, 1H), 4.92 (d, J=3.9Hz, 1H), 4.70 (d, J=8.5Hz, 1H), 4.54-4.51 (m, 3H), 4.40- 4.38 (m, 2H), 4.25-4.18 (m, 3H), 4.10-3.52 (m, 34H), 3.35-3.32 (t, J=8.6Hz, 1H), 2.77 (dd, J=12.5,4.6Hz, 1H), 2.03 (s, 6H), 1.80 (t, J=12.1Hz, 1H)

D-2.Neu5Gc_SSEA4- allyl (R₁=OH, R_N=NHGc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.99 (m, 1H), 5.38 (dd, J=1.2,17.2Hz, 1H), 5.29 (dd, J= 1.2,10.0Hz, 1H), 4.93 (d, J=4.0Hz, 1H), 4.69 (d, J=8.4Hz, 1H), 4.58-4.51 (m, 3H), 4.43- 4.37 (m, 2H), 4.28-4.17 (m, 3H), 4.14-3.52 (m, 34H), 3.33 (t, J=8.8Hz, 1H), 2.77 (dd, J= 4.8,12.4Hz, 1H), 2.03 (s, 3H), 1.81 (t, J=12.0Hz, 1H).

D-3.Ac- fluoride _ SSEA4- allyl (R₁=OH, R_N=NHCOCH₂F,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.96 (m, 1H), 5.36 (dd, J=1.6,17.2Hz, 1H), 5.25 (dd, J= 1.6,10.4Hz, 1H), 4.89 (d, J=46.4Hz, 2H), 4.88 (d, J=3.6Hz, 1H), 4.65 (d, J=8.4Hz, 1H), 4.53-4.45 (m, 3H), 4.39-4.32 (m, 2H), 4.22-3.51 (m, 37H), 3.30 (t, J=8.4Hz, 1H), 2.73 (dd, J=4.4,12.4Hz, 1H), 2.00 (s, 3H), 1.85 (t, J=12.4Hz, 1H).

D-4.Ac- phenyl _ SSEA4- allyl (R₁=OH, R_N=NHCOCH₂Ph,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.45-7.34 (m, 5H), 6.02 (m, 1H), 5.42 (dd, J=1.2,17.2Hz, 1H), 5.32 (dd, J=1.2,10.4Hz, 1H), 4.94 (d, J=4.0Hz, 1H), 4.72 (d, J=8.4Hz, 1H), 4.59- 4.52 (m, 3H), 4.46-4.38 (m, 2H), 4.30-3.50 (m, 38H), 3.42-3.32 (m, 4H), 2.77 (dd, J=4.4, 12.8Hz, 1H), 2.05 (s, 3H), 1.90 (t, J=12.0Hz, 1H).

D-5.Ac- repeatedly nitrogen base _ SSEA4- allyl (R₁=OH, R_N=NHCOCH₂N₃,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.95 (m, 1H), 5.35 (dd, J=1.6,17.2Hz, 1H), 5.25 (dd, J= 1.2,10.4Hz, 1H), 4.88 (d, J=3.6Hz, 1H), 4.65 (d, J=8.4Hz, 1H), 4.52-4.46 (m, 3H), 4.40- 4.32 (m, 2H), 4.23-4.18 (m, 3H), 4.12-3.50 (m, 36H), 3.30 (t, J=5.6Hz, 1H), 2.72 (dd, J= 4.8,12.8Hz, 1H), 2.00 (s, 3H), 1.84 (t, J=12.4Hz, 1H).

D-6.5'- repeatedly nitrogen base _ SSEA4- allyl (R₁=OH, R_N=N₃,R₁₀=OH)

¹HNMR(400MHz,D₂O): δ 5.99 (m, 1H), 4.40 (dd, J=1.6,17.2Hz, 1H), 5.29 (d, J= 10.4Hz, 1H), 4.92 (d, J=3.6Hz, 1H), 4.70 (d, J=8.4Hz, 1H), 4.56-4.51 (m, 3H), 4.43-4.38 (m, 2H), 4.26 (d, J=3.6Hz, 2H), 4.22 (d, J=6.4Hz, 1H), 4.10-3.46 (m, 35H), 3.36-3.32 (m, 1H), 2.74 (dd, J=4.8,12.4Hz, 1H), 2.04 (s, 3H), 1.79 (t, J=12.4Hz)；HRMS(ESI-TOF,M-H-) C₄₄H₇₁N₄O₃₃Calculated value 1183.4001, experiment value 1183.4056.

D-7.9'- repeatedly nitrogen base _ SSEA4- allyl (R₁=OH, R_N=NHAc, R₁₀=N₃)

¹H NMR(400MHz,D₂O) δ 5.96 (m, 1H), 5.36 (dd, J=1.6,17.3Hz, 1H), 5.26 (dd, J= 1.6,10.4Hz, 1H), 4.90 (d, J=3.6Hz, 1H), 4.68 (d, J=8.4Hz, 1H), 4.55-4.47 (m, 3H), 4.41- 4.35 (m, 2H), 4.25-4.14 (m, 3H), 4.10-3.41 (m, 34H), 3.31 (t, J=6.8Hz, 1H), 2.72 (dd, J= 4.8,12.8Hz, 1H), 2.02 (s, 3H), 1.79 (t, J=12.0Hz, 1H).

D-8.NHBz_SSEA4- allyl (R₁=OH, R_N=NHBz, R₁₀=OH)

¹H NMR(400MHz,D₂O)δ7.76-7.73(m,2H),7.59(m,1H),7.51-7.46(m,2H),5.90(m, 1H), 5.30 (dd, J=1.6,17.2Hz, 1H), 5.25 (dd, J=1.6,10.8Hz, 1H), 4.89 (d, J=3.6Hz, 1H), 4.67 (d, J=8.8Hz, 1H), 4.55-4.45 (m, 3H), 4.39-4.38 (m, 2H), 4.24-3.50 (m, 34H), 3.30 (t, J =8.0Hz, 1H), 2.77 (dd, J=4.4,12.4Hz, 1H), 2.01 (s, 3H), 1.90 (t, J=12.4Hz, 1H).

E:SSEA4 analog-aldehyde oxidation reaction

Process 6.

In certain exemplary embodiments, make SSEA4 analog-allyl based on the agitated solution in methanol and water smelly It is decomposed 15 minutes under carrier of oxygen atmosphere, in -70 DEG C of progress ozone.Pass through methyl sulfide (Me₂S) quenching reaction mixture, then very Sky evaporation solution.That aldehyde then passes through G15 to wanted SSEA4 analog-is column purified.(process 6)

SSEA4 analog-aldehyde¹H NMR

E-1:SSEA4- aldehyde (R_N=NHAc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.19 (t, J=4.9Hz, 1H), 4.89 (d, J=3.9Hz, 1H), 4.66 (d, J= 8.4Hz, 1H), 4.54-4.45 (m, 3H), 4.36 (t, J=6.5Hz, 1H), 4.25-4.20 (m, 1H), 4.15 (d, J= 3.1Hz, 1H), 4.08-3.47 (m, 32H), 3.37-3.30 (m, 1H), 2.73 (dd, J=12.4,4.6Hz, 1H), 2.00 (d, J =0.9Hz, 6H), 1.76 (t, J=12.1Hz, 1H).

E-2:Neu5Gc_SSEA4- aldehyde (R_N=NHGc, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.20 (t, J=4.9Hz, 1H), 4.91 (d, J=3.9Hz, 1H), 4.68 (d, J= 8.5Hz, 1H), 4.52 (dt, J=8.5,4.5Hz, 3H), 4.38 (t, J=6.5Hz, 1H), 4.27-4.22 (m, 1H), 4.17 (d, J=3.1Hz, 1H), 4.13-3.51 (m, 34H), 3.38-3.32 (m, 1H), 2.76 (dd, J=12.4,4.6Hz, 1H), 2.02 (s, 3H), 1.80 (t, J=12.1Hz, 1H).

E-3:Ac- fluoride _ SSEA4- aldehyde (R_N=NHCOCH₂F,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.21 (t, J=4.9Hz, 1H), 4.90 (d, J=46.4Hz, 2H), 4.69 (d, J= 8.5Hz, 1H), 4.52 (t, J=8.0Hz, 3H), 4.38 (t, J=6.4Hz, 1H), 4.24 (d, J=3.1Hz, 1H), 4.17 (d, J=3.2Hz, 1H), 4.10-3.45 (m, 33H), 3.40-3.32 (m, 1H), 2.78 (dd, J=12.4,4.6Hz, 1H), 2.03 (s, 3H), 1.81 (t, J=12.2Hz, 1H).

E-4:Ac- phenyl _ SSEA4- aldehyde (R_N=NHCOCH₂Ph,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.48-7.27 (m, 5H), 5.22 (t, J=4.9Hz, 1H), 4.92 (d, J= 4.0Hz, 1H), 4.69 (d, J=8.4Hz, 1H), 4.56-4.49 (m, 3H), 4.39 (t, J=6.5Hz, 1H), 4.26 (m, 1H), 4.18 (m, 1H), 4.10-3.45 (m, 34H), 3.43-3.34 (m, 1H), 2.76 (dd, J=12.4,4.6Hz, 1H), 2.03 (s, 3H), 1.78 (t, J=12.3Hz, 1H).

E-5:Ac- repeatedly nitrogen base _ SSEA4- aldehyde (R_N=NHCOCH₂N₃,R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 5.20 (t, J=4.9Hz, 1H), 4.90 (d, J=3.9Hz, 1H), 4.68 (d, J= 8.5Hz, 1H), 4.54-4.48 (m, 3H), 4.38 (t, J=6.4Hz, 1H), 4.24 (d, J=3.1Hz, 1H), 4.17 (d, J= 3.1Hz, 1H), 4.13-3.51 (m, 34H), 3.39-3.32 (m, 1H), 2.75 (dd, J=12.4,4.6Hz, 1H), 2.02 (s, 3H), 1.79 (t, J=12.2Hz, 1H).

E-6:9'- repeatedly nitrogen base _ SSEA4- aldehyde (R_N=NHAc, R₁₀=N₃)

¹H NMR(400MHz,D₂O) δ 5.20 (t, J=4.9Hz, 1H), 4.91 (d, J=3.9Hz, 1H), 4.69 (d, J= 8.5Hz, 1H), 4.52 (t, J=8.0Hz, 3H), 4.38 (t, J=6.4Hz, 1H), 4.24 (d, J=3.1Hz, 1H), 4.17 (d, J=3.2Hz, 1H), 4.10-3.45 (m, 32H), 3.39-3.32 (m, 1H), 2.74 (dd, J=12.5,4.6Hz, 1H), 2.03 (d, J=2.1Hz, 6H), 1.77 (t, J=12.1Hz, 1H).

E-7:NHBz_SSEA4- aldehyde (R_N=NHBz, R₁₀=OH)

¹H NMR(400MHz,D₂O) δ 7.83-7.76 (m, 2H), 7.63 (t, J=7.3Hz, 1H), 7.53 (t, J= 7.7Hz, 2H), 5.21 (t, J=4.9Hz, 1H), 4.92 (d, J=3.8Hz, 1H), 4.70 (d, J=8.5Hz, 1H), 4.57- 4.49 (m, 3H), 4.39 (t, J=6.5Hz, 1H), 4.26 (d, J=3.1Hz, 1H), 4.19 (d, J=3.3Hz, 1H), 4.16- 3.52 (m, 32H), 3.40-3.34 (m, 1H), 2.82 (dd, J=12.4,4.6Hz, 1H), 2.04 (d, J=4.7Hz, 3H), 1.87 (t, J=12.1Hz, 1H).

Example 3: it is crosslinked via sulfo group-EMCS to synthesize SSEA3/SSEA4 analog-CRM197 conjugate

Process 7

Conventional method:

Step A. by SSEA3 analog-amino or SSEA4 analog-it is amido modified at SSEA3 analog-mercaptan or SSEA4 analog-mercaptan

In order to synthesize SSEA3/4 analog CRM197 conjugate, the SSEA3/4 analog for blocking amine is connect with DTSSP Symbol reacts at room temperature in PBS buffer solution (pH 7.4).Solution ph is monitored by pH paper；When solution becomes slant acidity, Some sodium hydroxide solutions are added into reaction solution.After reactant being stirred at room temperature 12 hours, at room temperature to solution Middle addition DTT.Solution is kept stirring at 40 DEG C, then removes solvent by being concentrated under reduced pressure.It is pure finally by LH-20 col-umn chromatography Change residue, obtains SSEA3/4 analog-mercaptan.

Step B: CRM197 is modified to CRM197- maleimide.

Via the salt for repeatedly adding water and the commercially available CRM197 (1.0mg) of (Amicon Ultra-0.5,10kDa) removal of dialysing Later, residue is dissolved in PBS buffer solution (pH 6.5,1.0mL) and is transferred in sample flasket.Sulphur is added into solution Base-EMCS (1.0mg, 8.22 × 10^-6Mol), then reactant is kept stirring 2 hours at room temperature.Pass through Amicon Ultra- 0.5 (10kDa) purified mixture.Using MALDI-TOF inspection molecular weight and with after BCA analytical calculation protein content, incite somebody to action CRM197- maleimide is stored in PBS buffer solution (pH 7.2,1.0mg/mL) for next step.According to MALDI-TOF data can calculate the amount of maleimide functional group.For example, when CRM197- maleimide Molecular weight when being 61841, maleimide functional group's number on CRM197- maleimide is (61841- 58326)/193=18.2.

Step C: synthesis SSEA3/4 analog-CRM197 conjugate

CRM197- maleimide is dissolved in PBS buffer solution (pH 7.2, concentration 1.0mg/mL), then Different amounts of SSEA3/4 analog-mercaptan (5.0mg/mL, in PBS buffer solution, pH 7.2) is added into solution.In room temperature Lower stirring mixture 2 hours.SSEA3/4 analog-CRM197 conjugate is purified using Amicon Ultra-0.5 (10kDa), To remove unreacted SSEA3/4 analog-mercaptan and sodium ascorbyl phosphate via dialysis.Gained SSEA3/4 analog-CRM197 Conjugate can be analyzed by MALDI-TOF and be characterized to measure carbohydrate merger ratio.It is reacted with DTT and passes through LH-20 After col-umn chromatography purifying, unreacted SSEA3/4 analog-mercaptan can be recycled.

Table 1. engages number with the carbohydrate in CRM-197 conjugate via the SSEA4 analog that sulfo group-EMCS is generated

Example 4: it is crosslinked via sulfo group-EMCS to synthesize SSEA4-Gc-CRM197 conjugate

Process 8.

Step A: SSEA4-Gc- is amido modified for SSEA4-Gc- mercaptan

At room temperature, by DTSSP (5.0mg, 8.22 × 10^-6Mol) be added to containing SSEA4-Gc- amino (5.0mg, 4.01×10^-6Mol) in the flask in PBS buffer solution (pH 7.4,1.0mL).Solution ph is monitored by pH paper, works as solution When becoming slant acidity, sodium hydroxide solution (1 mole concentration/water) is added into solution into reaction solution.It is stirred at room temperature After reactant 12 hours, at room temperature into solution add DTT (5.0mg, 32.41 × 10^-6mol).Solution is kept at 40 DEG C Stirring 1 hour then removes solvent by being concentrated under reduced pressure.Residue is purified finally by LH-20 col-umn chromatography, is obtained SSEA4-Gc- mercaptan (5.0mg, 93%).

Step B: CRM197 is modified to CRM197- maleimide.

It is removed in commercially available CRM197 (1.0mg) via water and dialysis (Amicon Ultra-0.5,10kDa) is repeatedly added Salt after, residue is dissolved in PBS buffer solution (pH 6.5,1.0mL) and is transferred in sample flasket.Add into solution Add sulfo group-EMCS (1.0mg, 8.22 × 10^-6Mol), then reactant is kept stirring 2 hours at room temperature.Pass through Amicon Ultra-0.5 (10kDa) purified mixture.Using MALDI-TOF check molecular weight and with BCA analytical calculation albumen quality it Afterwards, CRM197- maleimide storage in PBS buffer solution (pH 7.2,1.0mg/mL) is used for next step.According to MALDI-TOF data can calculate the amount of maleimide functional group.For example, when CRM197- maleimide Molecular weight when being 61841, maleimide functional group's number on CRM197- maleimide is (61841- 58326)/193=18.2.

CRM197- maleimide is dissolved in PBS buffer solution (pH 7.2, concentration 1.0mg/mL), then Different amounts of SSEA4Gc- mercaptan (5.0mg/mL, in PBS buffer solution, pH 7.2) is added into solution.It is stirred at room temperature Mixture 2 hours.SSEA4-Gc-CRM197 conjugate is purified using Amicon Ultra-0.5 (10kDa), to come via dialysis Remove unreacted SSEA4-Gc- mercaptan and sodium ascorbyl phosphate.Gained SSEA4-Gc-CRM197 conjugate can pass through MALDI-TOF Analysis is to characterize to measure carbohydrate merger ratio, as shown in table 2.It reacts with DTT and is purified by LH-20 col-umn chromatography Later, unreacted SSEA4-Gc- mercaptan can be recycled.

Step C: the unreacted maleimide in CRM197- maleimide is captured

SSEA4-Gc-CRM197 conjugate is dissolved in PBS buffer solution (pH 7.2, concentration 1.0mg/mL) and Xiang Rong The 2 mercapto ethanol (5mg/mL, PBS buffer solution, pH 7.2) of 10.0 equivalents is added in liquid.It is small that mixture 2 is stirred at room temperature When.SSEA4-Gc-CRM197 conjugate is purified using Amicon Ultra-0.5 (10kDa), to remove unreacted via dialysis 2 mercapto ethanol and sodium ascorbyl phosphate, then freeze-drying be white powder.

Table 2

The combination of CRM197 and SSEA4-Gc

Example 5: the SSEA4 analog-CRM197 conjugate generated via SBAP crosslinking

Process 9.

CRM197 is dissolved in 0.1M phosphate buffer pH 7.4 (about 1mg/mL), and adds SBAP into solution (1.0mg).Agitating solution 2 hours at room temperature light and slowly.Mixture is then diluted with PBS buffer solution and passes through Amicon Ultra-0.5 (10kDa, 2X) centrifugal concentrating, is subsequently added into 0.1M phosphate buffer into concentrate and is centrifuged, be repeated 5 times. The modified CRM-197 of gained can by MALDI-TOF (cation mode, matrix are sinapic acid, water) analyze be characterized with Measure SBAP merger ratio.

Modified CRM197 is dissolved in 0.1M phosphate buffer pH 8.0 (about 1mg/mL), and is added into solution Add SSEA4- mercaptan analog.Mixture is stirred at room temperature 1 day.Mixture is then diluted and is passed through with PBS buffer solution Amicon Ultra-0.5 (10kDa) centrifugal concentrating, is subsequently added into 0.1M phosphate buffer into concentrate and is centrifuged, and repeats 5 times.Gained glycan-protein conjugate can be analyzed by MALDI-TOF (cation mode, matrix are sinapic acid, water) to be subject to Characterization is to measure carbohydrate merger ratio.(process 9)

Table 3. is via the carbohydrate merger ratio in the SBAP SSEA4 analog generated and CRM197 conjugate

Example 6: the SSEA4 analog-CRM197 conjugate generated via reduction amination crosslinking

Process 10

In certain embodiments, CRM197 is dissolved in 0.1M phosphate buffer (pH 6-9, about 1mg/mL), and to Enough SSEA4- aldehyde analog and sodium cyanoborohydride are added in solution.Agitating solution 3 days at room temperature light and slowly.Mixture Then it is diluted with deionized water and (10kDa centrifugal concentrating is subsequently added into 0.1M phosphate-buffered by Amicon Ultra-0.5 Liquid is into concentrate and is centrifuged, and is repeated 5 times.Gained glycan-protein conjugate passes through MALDI-TOF (cation mode, matrix For sinapic acid and water) analysis characterized to measure carbohydrate and engage number.(process 10)

Table 4: number is engaged with the carbohydrate in CRM197 conjugate via the SSEA4 analog that reduction amination generates

The immunoassays of example 7:SSEA4 analog-CRM197 conjugate

Exemplary methods

In order to prove SSEA4 analog-CRM197 conjugate (the effect of S1 to S10)/immunogenicity, female C57BL/6 0.5 μ g SSEA4 analog CRM197 conjugate (being applied in combination with 2.0 μ g glycolipid adjuvants) of mouse (each group n=5) intramuscular inoculation. Control mice is given only phosphate buffered saline and 2.0 μ g glycolipid adjuvants.Vaccine inoculation 2 months, every biweekly and often The antiserum for collecting immune mouse latter week of secondary vaccine inoculation.It is directed to using the fixed 96 holes titration disk inspection of SSEA4 The antibody titer of SSEA4.Enzyme linked immunosorbent absorption method (ELISA) is carried out using the fixed 96 holes titration disk of SSEA4.Letter speech It, diluted antiserum is cultivated 2 hours at room temperature together with fixed SSEA4.After wash cycle, then use Anti-igg or IgM specific antibody that HPR is combined detect the anti-SSEA4 antibody captured.

In order to determine whether glycan-protein combination method can interfere immune response, make primary SSEA4 and CRM197 via EMCS connector (M1), SBAP connector (S1) or reductive amination method (R1) are combined and are ground for immunogenicity as described above Study carefully.

Representative result

After four immunity inoculations, primary SSEA4 and all eight kinds of SSEA4 analogs are worked as and Gal-C34 adjuvant combination The IgG (Fig. 3 A) and IgM (Fig. 3 B) antibody for SSEA4 can be energetically induced when use.In different analog groups, resist The potency of SSEA4IgG and IgM antibody is without significant difference.In addition, Glc-C34, which works as, is total to administration with primary SSEA4 and other analogs When, it also can be used as induction for the IgG (Fig. 4 A) of SSEA4 and the vaccine adjuvant of IgM (Fig. 4 B).

In addition, the result shows that glycan-protein combination method can influence to be immunoreacted shown in Fig. 5.With Gal-C34 group It closes in use, the anti-SSEA4IgG antibody titer that SSEA4-EMCS-CRM197 (M1) induces is higher than SSEA4-SBAP-CRM197 (S1) and SSEA4-CRM197 (being combined via reductive amination method, R1).

The immunogenicity research of example 8:SSEA4 analog CRM197 conjugate

In order to prove the immunogenicity of SSEA4 analog CRM197 conjugate, five female BAl BIcs/c mouse intramuscular immunisation 2 μ g SSEA4 analog-CRM197 conjugates and 2 μ g glycolipid adjuvant C34 are inoculated with three times, every biweekly.In previous research, In the case where independent SSEA4 analog-protein conjugate without any adjuvant, anti-GloboH antibody titer is lower.Third Ten days after secondary immunity inoculation, obtain the antiserum from each immunogene and for the glycan containing 94 kinds of chemical synthesis glycan it is micro- Array is tested, including ball series glycan and other tumour related carbohydrate antigens.Due to carrying out someization to glycan Modification is learned, therefore in glycan array includes some functionality connectors to check cross reactivity.

The antibody that SSEA4-Gc-CRM197 conjugate is induced is by SSEA4-Gc, primary SSEA4 or tetra- bglii fragment of SSEA4 Specific recognition, but not by other TACA and functionality connector specific recognition.The high IgG of Serum-induced obtained from glycoconjugate Antibody titer shows that T cell dependent immunity is reacted.It is interesting that not observed for SSEA4-Gc or primary SSEA4 It is generated to significant IgM.About the IgG content for being directed to GloboH, the antibody titer that SSEA4-Gc CRM197 is induced is than natural shape The primary SSEA4-CRM197 conjugate of formula is much higher.Wherein, the conjugate of 6.9 SSEA4-Gc molecules and 1 CRM197 molecule It can induce highest antibody titer.

Mouse dose and immunity inoculation time-histories

In order to compare the immunogenicity of SSEA4 analog CRM197, ten groups of five mouse (8 week old female BAl BIcs/c mouse, BioLASCO, Taiwan) intramuscular immunization glycolipid C34.Immunity inoculation three times, every once every 2 weeks.Each vaccine contains 2 μ g SSEA4 Analog and 2 μ g C34.Control mice injects phosphate buffered saline (PBS).It is (immune before the inoculation of mouse first immunisation Before) and third time immunity inoculation after bloodletting in 10 days.10 minutes are centrifuged by 4,000 × g to obtain all serum.Pass through glycan Micro- array analyzes seroreaction.

Use the serological analysis of glycan array

Mouse blood is diluted with 1%BSA/PBST buffer (PBST buffer: PBS and 0.05%Tween-20, pH 7.4) Clearly.The micro- array of glycan blocks buffer (Pierce) to block 1 hour and delayed before the use with PBST at 4 DEG C with Superblock Fliud flushing is washed three times.Then serum dilution is introduced into the micro- array of glycan and is cultivated 1 hour at 4 DEG C.It is anti-to wash away excessive serum The mountain that body and micro- array are individually combined with the goat anti-mouse IgG antibody combined of Alexa Fluor 647 or DyLight 649 Sheep anti-Mouse IgM antibody is cultivated 1 hour at 4 DEG C in the dark together as secondary antibodies.Slide glass then with PBST wash three times and With micro- array Fluorescence chip reader (GenePix 4300A；Molecular Devices Corporation) in 635nm wave Long lower scanning and the image through scanning with GenePix Pro-6.0 analyze software (Axon Instruments, Union City, CA, USA) it is analyzed.

Other embodiments

All features disclosed in this specification can combine in any combination.Each feature disclosed in this specification can Through being replaced for identical, equivalent or similar purpose alternative characteristics.Therefore, unless explicitly stated otherwise, otherwise revealed Each feature is only an example of a series of equivalent or similar generic features.As described above, those who familiarize themselves with the technology can hold Easily determine the essential characteristic of the embodiment, and without departing from their spirit and scope in the case where can make various changes to embodiment Change and modifies to adapt it to various uses and condition.Therefore, other embodiments also belong in claim.

Claims (42)

1. a kind of immunogenic composition, it includes:

It (a) include carrier and the glycan conjugate of one or more glycan；And it is optionally existing

(b) adjuvant；

Wherein for each of one or more described glycan via connector in conjunction with the carrier, the conjugate has formula (III):

Wherein:

X₁For-OR or-SR, wherein R is oxygen or sulfur protecting group, the C being optionally substituted_1-10Alkyl, the virtue being optionally substituted Base, the acyl group being optionally substituted or the acylimino being optionally substituted；

R¹、R²、R³、R⁴、R⁵And each example of L independently selected from hydrogen, halogen, optionally be substituted alkyl, be optionally substituted Alkenyl, the alkynyl, the heterocycle being optionally substituted, the aryl ,-N that are optionally substituted that are optionally substituted₃、-NO₂、-N (R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

Wherein:

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；

R⁶Selected from hydrogen, halogen, optionally be substituted alkyl, optionally be substituted alkenyl, optionally be substituted alkynyl, view The aryl ,-N that situation is substituted₃、-NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C (O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

Wherein:

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；Or

R⁶For-OR^A；Wherein R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, The alkynyl being optionally substituted and the aryl being optionally substituted；

And

Its restrictive condition is that the glycan conjugate does not have formula (III-a) and (III-b):

2. immunogenic composition as claimed in claim 1, wherein L is-OH.

3. immunogenic composition as claimed in claim 2, wherein R¹、R²、R³、R⁴、R⁵And R⁶At least one example be-N₃。

4. immunogenic composition as claimed in claim 2, wherein R¹、R²、R³、R⁴、R⁵And R⁶At least one example be-F.

5. immunogenic composition as claimed in claim 1, wherein L has following formula:

Wherein:

R⁸、R⁹、R¹⁰And R¹¹Each example independently selected from hydrogen, halogen, the optionally alkyl, the optionally alkene that is substituted that are substituted Base, the alkynyl being optionally substituted, the heterocycle being optionally substituted, the aryl ,-N being optionally substituted₃、-NO₂、-N (R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；Wherein R¹²For H, OH or halogen；

R_NIt is selected from-N₃、-NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C (O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted.

6. immunogenic composition as claimed in claim 5, wherein the glycan conjugate has formula (IV):

7. immunogenic composition as claimed in claim 6, wherein R¹、R²、R³、R⁸、R⁹、R¹⁰、R¹¹And R_NAt least one example be- N₃。

8. immunogenic composition as claimed in claim 6, wherein R¹、R²、R³、R⁸、R⁹、R¹⁰、R¹¹And R_NAt least one example be- F。

9. such as the immunogenic composition of any one of claims 1 to 8, wherein the carrier be protein, it is lipid, lipidization Protein, virus, peptide or glycopeptide dendritic.

10. immunogenic composition as claimed in claim 9, wherein the carrier is the protein selected from the group being made up of: Tetanus toxoid, diphtheria toxoid, diphtheria toxin cross reacting material 197, the segment C of TT, keyhole spiral shell blood cyanogen albumen, ox blood Pure albumen, 3-protein d, outer membrane protein and pneumolysin.

11. being made of wherein the carrier protein matter is selected from TT, DT and CRM197 such as the immunogenic composition of claim 10 Group.

12. such as the immunogenic composition of claim 11, wherein the glycan conjugate has formula (IV-a) or (IV-b):

Wherein m is integer 1 to 40, including endpoint.

13. such as the immunogenic composition of any one of claims 1 to 8, wherein the connector is Heterobifunctional connector Or bifunctional connector.

14. such as the immunogenic composition of any one of claims 1 to 8, wherein the adjuvant is can be thin in conjunction with dendron shape The glycolipid of CD1d molecule on born of the same parents.

15. as any one of claims 1 to 8 immunogenic composition, wherein the adjuvant be C34,7DW8-5, C17, C23, Glc-C34, aluminium salt, squalene, MF59 or QS-21.

16. such as the immunogenic composition of any one of claims 1 to 8, wherein the immunogenic composition can induce For the immune response of cancer cell.

17. wherein the cancer cell is selected from the group being made up of: the cancer of the brain is thin such as the immunogenic composition of claim 16 Born of the same parents, lung carcinoma cell, breast cancer cell, cancer cell of oral cavity, esophageal cancer cell, stomach cancer cell, liver cancer cells, cholangiocarcinoma cell, pancreas Cancer cell, colon cancer cell, renal carcinoma cells, bone cancer cells, skin cancer cell, cervical cancer cell, ovarian cancer cell and preceding Column adenocarcinoma cell.

18. such as the immunogenic composition of claim 16, wherein the immune response includes being specifically bound to one or more The generation of the antibody of antigen selected from the group being made of SSEA3 and SSEA4.

19. such as the immunogenic composition of claim 18, wherein in these antibody and on cancer cell or cancer stem cell surface One or more of expressed SSEA3 and SSEA4 antigen.

20. wherein these antibody include IgG antibody such as the immunogenic composition of claim 18.

21. a kind of cancer vaccine, it includes the immunogenic compositions of therapeutically effective amount such as any one of claims 1 to 15 And pharmaceutically acceptable excipient.

22. such as the cancer vaccine of claim 21, wherein the cancer vaccine can induce the antitumor immune reaction of individual.

23. a kind of immunogenic composition such as any one of claims 1 to 15 or the cancer epidemic disease such as claim 21 or 22 The purposes of seedling is used to prepare the medicament for treating the cancer of individual in need.

24. such as the purposes of claim 23, wherein the medicament and another therapeutic agent group amount to administration.

25. such as the purposes of claim 23, wherein the cancer is selected from the group being made up of: the cancer of the brain, lung cancer, breast cancer, oral cavity Cancer, cancer of the esophagus, gastric cancer, liver cancer, cholangiocarcinoma, cancer of pancreas, colon cancer, renal cancer, osteocarcinoma, cutaneum carcinoma, cervix cancer, oophoroma and Prostate cancer.

26. such as the purposes of claim 25, wherein the cancer cell of the cancer expresses SSEA3 and/or SSEA4 on cell surface Antigen.

27. such as the purposes of claim 23, wherein the individual is the mankind.

28. such as the purposes of claim 23, wherein the medicament is subcutaneous administration.

29. a kind of separated monoclonal antibody and/or its binding fragment are for IMMUNOGENIC COMPOSITION as claimed in claim 1 Object and generate.

30. a kind of composition it includes a effective amount of antibody such as claim 29 or antigen-binding fragment and pharmaceutically may be used The carrier of receiving.

31. a kind of method for being used to prepare immunogenic composition as claimed in claim 1.

32. wherein the method includes such as the method for claim 31:

Carrier is provided；

Make one or more glycan in conjunction with the carrier by association reaction；

Wherein each of one or more described glycan are S SEA3 or SSEA4.

33. a kind of immunogenic composition, it includes the multivalence of one or more of targeting SSEA4 and SSEA3 and the like Construct, wherein these glycan are connected to template；And carrier,

Wherein n can be integer 1 to 10；

Wherein shown in the formula III of the structure of glycan as described in claim 1；

If wherein n be 2 or be greater than 2, each glycan can on another glycan or aspartyl peptide on aspartyl peptide Different glycan is identical.

34. such as the composition of claim 33, wherein the glycan is selected from the group being made of SSEA3 and SSEA4.

35. such as the composition of claim 33, wherein the multivalence construct has a structure that

The wherein R on each glycan moiety¹、R²、R³、R⁴、R⁵、R⁶And L can be identical or different.

36. a kind of compound, with formula (I):

Or its salt,

Wherein:

X₁For-OR or-SR, the C that wherein R is hydrogen, oxygen or sulfur protecting group, is optionally substituted_1-10Alkyl is optionally substituted Aryl, the acyl group being optionally substituted or the acylimino being optionally substituted；

R¹、R²、R³、R⁴、R⁵And each example of L independently selected from hydrogen, halogen, optionally be substituted alkyl, be optionally substituted Alkenyl, the alkynyl, the heterocycle being optionally substituted, the aryl ,-N that are optionally substituted that are optionally substituted₃、-NO₂、-N (R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

Wherein:

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；

R⁶Selected from hydrogen, halogen, optionally be substituted alkyl, optionally be substituted alkenyl, optionally be substituted alkynyl, view The aryl ,-N that situation is substituted₃、-NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C (O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

Wherein:

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；Or R⁶For-OR^A；Wherein R^AEach example it is independent Ground is selected from hydrogen, the alkyl being optionally substituted, the alkenyl being optionally substituted, the alkynyl that is optionally substituted and optionally through taking The aryl in generation；

And its restrictive condition is that the compound does not have formula (I-a) or (I-b):

37. wherein L is-OH such as the compound of claim 35.

38. wherein L has following formula such as the compound of claim 35:

Wherein:

R⁸、R⁹、R¹⁰And R¹¹Each example independently selected from hydrogen, halogen, the optionally alkyl, the optionally alkene that is substituted that are substituted Base, the alkynyl being optionally substituted, the heterocycle being optionally substituted, the aryl ,-N being optionally substituted₃、-NO₂、-N (R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C(O)R^A、-C(O)OR^A、-S(O)R^A、- SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；Wherein R¹²For H, OH or halogen；

R_NIt is selected from-N₃、-NO₂、-N(R^B)₂、-N(R^A)C(O)R^A、-OR^A、-OC(O)R^A、-SR^A、-C(O)N(R^B)₂、-CN、-C (O)R^A、-C(O)OR^A、-S(O)R^A、-SO₂R^A、-SO₂N(R^B)₂And-NHSO₂R^B；

R^AEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted；And

R^BEach example be substituted independently selected from hydrogen, optionally alkyl, be optionally substituted alkenyl, be optionally substituted Alkynyl, the heterocycle being optionally substituted and the aryl being optionally substituted.

39. such as the compound of claim 35, wherein the compound has formula (II):

40. such as the compound of claim 39, wherein R¹、R²、R³、R⁸、R⁹、R¹⁰And R¹¹At least one example be-F.

41. such as the compound of claim 39, wherein R¹、R²、R³、R⁸、R⁹、R¹⁰And R¹¹At least one example be-N₃。

42. a kind of purposes of such as compound of claim 36 or 39, is used to prepare treatment hyperproliferative disease or symptom Medicament.