CN106267231A - A kind of little molecule CD19 antibody polypeptides and the conjugate of cyclophosphamide coupling - Google Patents
A kind of little molecule CD19 antibody polypeptides and the conjugate of cyclophosphamide coupling Download PDFInfo
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- CN106267231A CN106267231A CN201610747982.XA CN201610747982A CN106267231A CN 106267231 A CN106267231 A CN 106267231A CN 201610747982 A CN201610747982 A CN 201610747982A CN 106267231 A CN106267231 A CN 106267231A
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- cyclophosphamide
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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Abstract
The invention discloses the coupling conjugate of little molecule CD19 antibody polypeptides and cyclophosphamide, belong to antibody coupling drug world.It is characterized in that: the coupling conjugate of cyclophosphamide and CD19 antibody polypeptides；Described CD19 antibody polypeptides is brand-new sequence.Coupling conjugate preparation process includes: (1) prepares the cyclophosphamide of 2 8mmol/L concentration with 0.1 mol/L HCl solution；(2) 4 DEG C, drip 1%NaNO2, continuously stirred, react 20 30 minutes；(3) with the CD19 antibody polypeptides described in buffer solution of pH8 10；(4) described CD19 antibody polypeptides solution adds diazotizing cyclophosphamide, regulate pH to 9 10；(5) 48 DEG C, react 2 hours；(6) PBS 2 days, to obtain final product.Described coupling conjugate, in preparation application in tumor.Have the beneficial effects that the tumor-targeting promoting cyclophosphamide, improve curative effect, reduce toxic and side effects.
Description
Technical field
The present invention is antibody coupling drug world.In particular it relates to CD19 antibody polypeptides coupling antineoplastic agent
The conjugate of thing cyclophosphamide.
Background technology
Non-Hodgkin lymphoma (NHL) is the general name with very strong one group of heterogeneous independent disease.Also it is ratio in China
A kind of tumor more typically, in common cancer ranks within first 10.NHL pathological changes be occur mainly in lymph node,
The lymphatic organ such as spleen, thymus, it is possible to occur to swell in the lymphoid tissue of outside lymph node and the pernicious of lymphohematological of organ
Tumor.It is classified as three basic forms of it: B cell, T cell and NK/T cell NHL according to cell derived.Clinical most of NHL are B
Cellular type, accounts for sum 70%～85%.NHL is layered the most more complicated in histological typing, clinical manifestation with treatment individuation,
But a kind of tumor that likely height is cured.At present sickness rate in all ages and classes stage in the trend substantially risen.
The most multiple multicenter, international random contrast clinical trial research datas prove, the first-line treatment scheme of its standard should
More manage for treatment non-Hodgkin lymphoma at present when being CHOP scheme (cyclophosphamide, doxorubicin, vincristine, prednisone)
The first-line treatment scheme thought.More than 50% non-Hodgkin′s is cured.While it is true, this therapeutic scheme can cause more serious poison
Side effect.Common adverse effect: 1. bone marrow depression；2. phlebitis；3. digestive tract reaction；4. alopecia etc..Wherein, the double merit of cyclophosphamide
Can alkylating agent and cell cycle nonspecific agent (CCNSA), may interfere with DNA and RNA function, especially with bigger on the former impact, it with
There is cross link, suppression DNA synthesis in DNA, acts on the most obvious to the S phase.It is clinically used for malignant lymphoma and has a certain curative effect, but ring
Phosphamide also has bigger toxic and side effects.Producing main reason is that of toxic and side effects, cyclophosphamide is that broad-spectrum anti-tumor is wanted
Asking, specificity is poor, also has certain lethal effect to normal cell.Therefore, inventor want to by cyclophosphamide with
Specific antibody coupling, thus produce the targeting of tumor cell, improve curative effect, reduce toxic and side effects.
CD19 is a kind of B cell specificity transferring film glycoprotein, is expressed in B system cell (not including mature plasme cell) and folliculus
In dendritic cell, CD19 is a kind of important signal transduction molecule, the growth activation of regulation bone-marrow-derived lymphocyte and activation, is adjusting
The signal threshold value of joint bone-marrow-derived lymphocyte antigen receptor or other surface receptors plays an important role, is and bone-marrow-derived lymphocyte differentiation, work
Change, propagation and antibody produce relevant important membrane antigen.B cell lymphoma cell surface receptor CD19 plays pivotal role.
CD19 antibody with targeting in B cell lymphoma cell surface, and itself can have the effect of lymphoma.But
The molecular weight being CD19 antibody is relatively big, has antigenicity, and the long-term people of being used for knows from experience generation and neutralizes reaction, reduces drug effect.Therefore, originally
Invention provides a kind of high specificity, little molecule CD19 antibody polypeptides that purity is higher and the conjugate of cyclophosphamide coupling, promotees
Enter the tumor-targeting of cyclophosphamide, improve curative effect, reduce toxic and side effects.
Summary of the invention
Goal of the invention
The present invention provides a kind of high specificity, the knot of the higher little molecule CD19 antibody polypeptides of purity and cyclophosphamide coupling
Compound, promotes the tumor-targeting of cyclophosphamide, improves curative effect, reduces toxic and side effects.
Technical scheme
Technical program of the present invention lies in the coupling conjugate providing a kind of little molecule CD19 antibody polypeptides with cyclophosphamide,
It is characterized in that: the coupling conjugate of cyclophosphamide and CD19 antibody polypeptides；Described CD19 antibody polypeptides is brand-new sequence.Described
The preparation of coupling conjugate, step includes: (1) prepares the cyclophosphamide of 2-8mmol/L concentration with 0.1mol/L HCl solution；
(2) 4 DEG C, drip 1%NaNO2, continuously stirred, react 20-30 minute；(3) with the CD19 described in buffer solution of pH8-10
Antibody polypeptides；(4) described CD19 antibody polypeptides solution adds diazotizing cyclophosphamide, regulate pH to 9-10；(5) 4-8 DEG C,
React 2 hours；(6) PBS 2 days, to obtain final product.Described CD19 antibody polypeptides uses chemical synthesis to prepare.Described coupling is tied
Compound, in preparation application in tumor.It is preferably in preparation for treating answering in malignant lymphatic tumor medicine
With, in particular for the application in treatment non-Hodgkin lymphoma medicine.
Beneficial effect
The little molecule CD19 antibody polypeptides of the present invention and the coupling conjugate of cyclophosphamide, can be used for promoting cyclophosphamide
Tumor-targeting, improves curative effect, reduces toxic and side effects.Have the beneficial effects that (1) improves cyclophosphamide tumor tissues in vivo
Concentration；Reduce cyclophosphamide in whole blood, the distribution of the tissues such as Yi Jixin, liver, spleen, lung, kidney, brain simultaneously；(2) significance suppression is swollen
The propagation of oncocyte；(3) toxicity of cyclophosphamide is reduced；(4) little molecule CD19 antibody polypeptides is combined with the coupling of cyclophosphamide
Thing can specific binding with tumor cell；(5) tumor-bearing mice survival rate is improved.
Detailed description of the invention
Polypeptide is by Shanghai raw work gill synthesis.
Embodiment 1
Prepare the coupling conjugate of CD19 antibody polypeptides and cyclophosphamide: prepare 2mmol/L with 0.1mol/L HCl solution
The cyclophosphamide of concentration；4 DEG C, drip 1%NaNO2, continuously stirred, react 20 minutes；Described in the buffer solution of pH8
CD19 antibody polypeptides；CD19 antibody polypeptides solution adds diazotizing cyclophosphamide, regulates pH to 9；At 4-8 DEG C, reaction 2 is little
Time；Use PBS 2 days again, lyophilizing, to obtain final product.
Embodiment 2
Prepare the coupling conjugate of CD19 antibody polypeptides and cyclophosphamide: prepare with 0.1mol/L HCl solution
The cyclophosphamide of 8mmol/L concentration；4 DEG C, drip 1%NaNO2, continuously stirred, react 30 minutes；With delaying of pH10
Rush the CD19 antibody polypeptides described in liquid dissolving；CD19 antibody polypeptides solution adds diazotizing cyclophosphamide, regulates pH to 10；
At 4-8 DEG C, react 2 hours；Use PBS 2 days again, lyophilizing, to obtain final product.
Embodiment 3
Employment B cell lymphoma Transplanted tumor model detection CD19 antibody polypeptides is internal with the coupling conjugate of cyclophosphamide
Vigor.
6-8w SCID mice in age, mice is randomly divided into 5 groups, male and female half and half, often group 10.(1) blank group；(2) conjugate
Low dose group；(3) dosage group in conjugate；(4) conjugate high dose group；(5) cyclophosphamide positive group.Set up human B cell to drench
Bar tumor Transplanted tumor model, the 7th day after inoculation lymphoma cell, tail intravenously administrable is administered respectively.Dosage regimen is: blank group
0.05,0.1,0.2mmol/Kg adding the solvent of same volume, experimental group is that conjugate sets 3 dosage:, and positive group is
The cyclophosphamide of 0.2mmol/Kg, is administered, continuous 14 days every day.After 21 days, observe mouse survival quantity, calculate survival rate.Knot
Fruit display, conjugate can protect tumor-bearing mice effectively, dosage 0.05,0.1,0.2mmol/Kg time can improve tumor-bearing mice
Survival rate, survival rate is respectively 67.43,78.67,79.03%.
Embodiment 4
The in-vitro multiplication rate experiment of B cell lymphoma: use MTT colorimetry.By thin for the human B cell lymphoma of logarithmic growth
Born of the same parents, with 1.0 × 105Add in 96 well culture plates, cultivate 24h；Experiment is divided into 5 groups, is respectively blank group, positive group and low middle height
The CD19 antibody polypeptides that the embodiment 1 of dosage obtains and the coupling conjugate of cyclophosphamide；It is separately added into the DMEM of same volume
Culture medium (HyClone, containing hyclone 20%), cyclophosphamide (1mmol/ml) and CD19 antibody polypeptides and the idol of cyclophosphamide
Connection compound (10,20,40 μm ol/ml).Every hole sets five multiple holes, cultivates 48h, and every hole adds MTT, after effect 4h, adds
DMSO, hatches 30min, measures absorbance A value at microplate reader 620nm, becomes B cell lymphoma suppression ratio=(1 reality by formula
Test group light absorption value/matched group light absorption value) × 100%.The B of the coupling conjugate experimental group of cyclophosphamide positive group and embodiment 1
The suppression ratio of cell lymphoma cell is respectively 78.9,47.02,69.1 and 87.2% (p ＜ 0.05).Result shows, embodiment 1
Coupling conjugate can effectively suppress growth of tumour cell, the tumor control rate of high dose conjugate be higher than cyclophosphamide.
Embodiment 5
Employment B cell lymphoma Transplanted tumor model detection CD19 antibody polypeptides is internal with the coupling conjugate of cyclophosphamide
Distribution.
6-8w SCID mice in age, mice is randomly divided into 3 groups, male and female half and half, often group 6.(1) blank group；(2) conjugate
Group；(3) cyclophosphamide positive group.Set up human B cell lymphoma Transplanted tumor model, the 7th day after inoculation lymphoma cell, point
Other tail intravenously administrable is administered.Dosage regimen is: blank group adds the solvent of same volume, and experimental group is 0.2mmol/Kg dosage
Conjugate, positive group is the cyclophosphamide of 0.2mmol/Kg, is administered every day, continuous 7 days.After 14 days, after sacrifice, respectively
Taking whole blood, Yi Jixin, liver, spleen, lung, kidney, brain, tumor etc. are organized.Whole blood anticoagulant heparin, by thin for heparin anti-coagulating centrifugation blood
Born of the same parents, take supernatant blood plasma 0.5ml, add 2.67%HCLO4, centrifugation plasma protein, supernatant heating in water bath 100 DEG C 20 minutes,
To be measured；Each tissue through homogenate, removing protein, be centrifuged after, take supernatant.The supernatant of blood plasma and tissue is added respectively NBP at alkalescence bar
Carry out chromogenic reaction under part, under 575nm wavelength, then carry out colorimetric determination.Calculate mice plasma and respectively organize cyclophosphamide
Concentration.
Table 1 conjugate is blood plasma and tissue distribution concentration in tumor model animal
* P < 0.05, * * P < 0.01 is compared with positive group.
Experimental result is shown in Table 1, compares with cyclophosphamide positive group, CD19 antibody polypeptides and the coupling conjugate of cyclophosphamide
Concentration in group in-vivo tumour tissue is significantly raised, (p ＜ 0.05), and the tissue such as the heart, liver, spleen, lung, kidney, brain, and in blood plasma
Concentration of cyclophosphamide substantially reduce, the especially drug level in blood plasma is 23.98 ± 32.62 μ g/ml (p ＜ 0.05), has
Significant difference.
Embodiment 6
The coupling conjugate of CD19 antibody polypeptides and cyclophosphamide toxic action.Use embodiment 1 conjugate, measure
Median lethal dose(LD 50): take 60 mices and be randomly divided into 6 groups, often group l0 is only.Each group mice elder generation fasting 12h, lumbar injection is different respectively
Embodiment 2 conjugate of dosage, dosage is respectively 125,250,500,1000,2000,5000mg/Kg, to after test medicine again
Fasting 3-4 hour.Hereafter Continuous Observation 2 weeks, with or without the phenomena of mortality, calculate median lethal dose(LD 50) LD50.And observe skin, mucosa,
Hair color, eyes, breathe, circulate, autonomous and central nervous system's behavior expression.
Result: embodiment 1 conjugate is 1743.98mg/Kg to median lethal dose(LD 50) LD50 of mice.
Embodiment 7
Conjugate and the combination rate of B cell lymphoma cell: use ELISA method, by the human B cell lymph of logarithmic growth
Oncocyte, with 1.0 × 105Add in 96 hole ELISA Plate, 4 DEG C, be coated overnight；After PBS washes plate tri-times, by the combination of embodiment 1
Thing, presses blank group, and Concentraton gradient 0,10,20,40 μm ol/ml is separately added in 96 orifice plates, and the most every hole adds quantitative resisting
CD19 antibody competition combines, 37 DEG C, after hatching 2 hours；After PBS washes plate tri-times, add coupling horseradish peroxidase
The anti-human IgG antibodies of (horseradish peroxidase, HRP), hatches 1 hour by 37 DEG C；After PBS washes plate tri-times again, add
ABC solution, hatches 10-20 minute, finally terminates reaction with flowing water；Microscope is observed, and described embodiment 1 conjugate is thin with tumor
The cell that born of the same parents combine is brownish black, for positive cell, uses computer software to calculate the quantity of the cell that is positive.
Result: embodiment 1 conjugate has significance poor with the quantity of B cell lymphoma Cell binding compared with blank group
Different, when dosage is 40 μm ol/ml, positive cell quantity reaches 475.9 ± 34.6 (p ＜ 0.05).As can be seen here, embodiment 1 is tied
Compound and B cell lymphoma Cell binding rate are with dosage escalation.
SEQUENCE LISTING
<110>Suzhou Pu Luoda bio tech ltd
<120>a kind of CD19 antibody polypeptides and the conjugate of cyclophosphamide coupling
<130>
<160> 1
<170> PatentIn version 3.3
<210> 14
<211> 44
<212> PRT
<213>artificial sequence
<400> 1
Ala Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser
1 5 10 15
Val Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro
20 25 30
Lys Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg
35 40
Claims (6)
1. one kind little molecule CD19 antibody polypeptides and the coupling conjugate of cyclophosphamide, it is characterised in that: cyclophosphamide and CD19
The coupling conjugate of antibody polypeptides；The sequence of described CD19 antibody polypeptides is SEQ ID NO:1.
The preparation of coupling conjugate the most according to claim 1, it is characterised in that: step includes: (1) uses 0.1 mol/L
The cyclophosphamide of HCl solution preparation 2-8mmol/L concentration；(2) 4 DEG C, dripping 1%NaNO2, continuously stirred, reaction 20-30 divides
Clock；(3) with the CD19 antibody polypeptides described in buffer solution of pH8-10；(4) described CD19 antibody polypeptides solution adds weight
The cyclophosphamide of nitridation, regulates pH to 9-10；(5) 4-8 DEG C, react 2 hours；(6) PBS 2 days, to obtain final product.
Coupling conjugate the most according to claim 2, it is characterised in that: described CD19 antibody polypeptides uses chemical synthesis
Preparation.
4. according to the arbitrary described coupling conjugate of claim 1-3, it is characterised in that: in preparation in tumor
Application.
Coupling conjugate the most according to claim 4, it is characterised in that: in preparation for treating in malignant lymphatic tumor medicine
Application.
Coupling conjugate the most according to claim 5, it is characterised in that: it is used for treating non-Hodgkin lymphoma medicine in preparation
In application.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080125A (en) * | 2010-07-02 | 2013-05-01 | 安吉奥开米公司 | Short and D-amino acid-containing polypeptides for therapeutic conjugates and uses thereof |
| CN104379169A (en) * | 2012-08-14 | 2015-02-25 | Ibc药品公司 | T-cell redirecting bispecific antibodies for treatment of disease |
| CN104758946A (en) * | 2015-04-07 | 2015-07-08 | 中国科学院过程工程研究所 | Antibody-coupled drug as well as preparation and application thereof |
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- 2016-08-29 CN CN201610747982.XA patent/CN106267231A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080125A (en) * | 2010-07-02 | 2013-05-01 | 安吉奥开米公司 | Short and D-amino acid-containing polypeptides for therapeutic conjugates and uses thereof |
| CN104379169A (en) * | 2012-08-14 | 2015-02-25 | Ibc药品公司 | T-cell redirecting bispecific antibodies for treatment of disease |
| CN104758946A (en) * | 2015-04-07 | 2015-07-08 | 中国科学院过程工程研究所 | Antibody-coupled drug as well as preparation and application thereof |
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