CN104707136A - Novel bifunctional antibody conjugate, preparation method and application thereof - Google Patents
Novel bifunctional antibody conjugate, preparation method and application thereof Download PDFInfo
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- CN104707136A CN104707136A CN201310697699.7A CN201310697699A CN104707136A CN 104707136 A CN104707136 A CN 104707136A CN 201310697699 A CN201310697699 A CN 201310697699A CN 104707136 A CN104707136 A CN 104707136A
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Abstract
The invention provides a novel bifunctional antibody conjugate, a preparation method and application thereof. The bifunctional antibody conjugate provided by the invention includes the structure of: a first antibody specifically directed at immune cells and a second antibody specifically directed at tumor markers, with the two antibodies coupled together through a coupling arm. The bifunctional antibody conjugate can significantly improve the tumor-killing activity after combination with an immune cell (like CIK). The invention also provides application of the novel bifunctional antibody conjugate in treatment of tumors and other diseases. The invention also provides an anti-HER2 positive cancer bispecific antibody and a preparation process. According to the invention, the bispecific antibody with high coupling rate and high activity can be simply and quickly produced, and the CIK tumor-killing activity can be improved by 2-6 times.
Description
Technical field
The present invention relates to biological medicine and antibody chemical coupling techniques field.More specifically, the present invention relates to New-type bifunctional antibody coupling matter and method for making thereof and purposes.
Background technology
According to World Health Organization's statistics, the number of malignant tumor is died from every year more than 7,000,000 in the whole world, will become the number one killer threatening human survival gradually.
Cellular immunotherapy is a kind of emerging, brand-new antitumour treatments with significant curative effect, has been acknowledged as a kind for the treatment of means active, the most rising in 21st century combined therapy of tumour pattern.
Bi-specific antibody is treated as the popular technology in one, current cell therapy field, combine the advantage of antibody and cell therapy, be applied to cancer, autoimmune disease and stem-cell therapy abroad, a part has entered clinical trial and has achieved significant antitumor efficacy.Bi-specific antibody utilizes antibody and antigenic specificity combination principle that lethal cell-targeting is positioned tumor cell position, and technology maturation is reliable, and side effect is little, high specificity, is the preferred option of cell therapy.
According to statistics, within 2010, global monoclonal antibody medicine market has reached the scale of 50,000,000,000 dollars, the total amount of transactions of 2009 ~ 2011 years bi-specific antibodys is also more than 3,500,000,000, infer and will occupy the larger market share afterwards than the better bi-specific antibody of monoclonal antibody medicine drug effect, become the biological medicine that sales volume is maximum.
At present, there is the bi-specific antibody multi-form more than 40 kinds to be synthesized at laboratory, and also had more bi-specific antibody in continuous synthesis.Existing more than ten plant bi-specific antibody and entered the clinical I phase or II, III phase is studied so far, wherein Herceptin/OKT3 bi-specific antibody has entered the clinical I/II phase and has studied, CD20/CD3 bi-specific antibody has entered the clinical I phase and has studied, bi-specific antibody " Catumaxomab " in 2009 by the medicine of European drug administration official approval as the pernicious ascites pleural fluid for the treatment of.
At present, along with going deep into gradually of research, bi-specific antibody is increasingly clear and definite at the mechanism of action for the treatment of and auxiliary treatment immune related diseases, there is significant curative effect, also the focus of antibody engineering research field has been become, therefore, bi-specific antibody has broad application prospects in the treatment of immune disease.
But up to the present, the chemical coupling efficiency of bi-specific antibody can't be satisfactory.Therefore, this area is in the urgent need to developing the technique of new production bi-specific antibody.
Summary of the invention
The object of the present invention is to provide a kind of newly, the method for coupling efficiency is high, method is easy production bi-specific antibody.
In a first aspect of the present invention, provide a kind of 1. 1 kinds of bifunctional antibody conjugates, described bifunctional antibody conjugate has structure described in formula Ia or formula Ib:
A-B-C (Ia), or
C-B-A (Ib)
Wherein,
A is the first antibody of specificity for immunocyte;
B is the coupling arm between A and C;
C is the second antibody of specific for cancer mark;
"-" represents the chemical bond connecting said elements.
In another preference, described first antibody is selected from lower group: the antibody of AntiCD3 McAb, anti-CTLA 4 antibody or its combination; And/or
Described second antibody is selected from lower group: the antibody of anti-HER2, the antibody of anti-EGFR or its combination.
In another preference, described first antibody and/or second antibody are monoclonal antibodies.
In another preference, described antibody coupling matter has one or more characteristics following:
A) can simultaneously with CIK cell and these two kinds of Cell bindings of tumor cell;
B) can (6,12,24 months or longer) preserve for a long time under 4 DEG C or-20 DEG C of conditions.
In another preference, described coupling arm is selected from lower group:
In formula, "-" of each structural formula both sides represents the key (chemical bond or covalent bond) be connected with second antibody with first antibody respectively.
In another preference, described bifunctional antibody conjugate structure is as follows:
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody;
And-the HN-be connected with X with Z or-NH-represents the-NH derived from antibody
2the imido grpup of group.
Bifunctional antibody conjugate as described in the first aspect of the invention, described first antibody is anti-cd 3 antibodies, and described second antibody is Anti-HER 2.
In second aspect present invention, provide a kind of pharmaceutical composition, described compositions contains the bifunctional antibody conjugate described in first aspect present invention, and pharmaceutically acceptable carrier.
In a third aspect of the present invention, provide a kind of method of pharmaceutical compositions, comprise step: the bifunctional antibody conjugate described in first aspect present invention is mixed with pharmaceutically acceptable carrier, thus obtained pharmaceutical composition.
In a fourth aspect of the present invention, provide a kind of method preparing antitumor drug, comprise step:
A bifunctional antibody conjugate described in first aspect present invention mixes with immunocyte by (), thus form the complex of " bifunctional antibody conjugate-immunocyte ",
B described complex is made antitumor drug by ().
In another preference, described medicine is liquid form.
The purposes of bifunctional antibody conjugate as described in the first aspect of the invention, for the preparation of the medicine of disease therapy.
In another preference, described disease comprises tumor.
In a fifth aspect of the present invention, provide a kind of method producing bifunctional antibody conjugate described in first aspect present invention, it comprises step:
A () provides the first intermediate shown in formula II; With the second intermediate shown in formula III
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody;
B the second intermediate shown in the first intermediate shown in formula II and formula III is carried out coupling reaction by (), thus form the bifunctional antibody conjugate shown in formula IV:
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody.
In another preference, described method comprises:
(1) monoclonal antibody of AntiCD3 McAb is dissolved in reaction buffer A; Traut ' s reagent is dissolved in ddH
2o is mixed with mother solution, with reaction buffer A or ddH during use
2o dilutes.
(2) monoclonal antibody of anti-HER2 is dissolved in reaction buffer B; Sulfo-SMCC is dissolved in ddH
2o is mixed with mother solution, with reaction buffer B or ddH during use
2o dilutes.
(3) mixed with mol ratio 5:1-10:1 with CD 3-resisting monoclonal antibody by Traut ' s reagent, Sulfo-SMCC mixes with mol ratio 4:1-20:1 with anti-HER 2 monoclonal antibody.
(4) reactant after mixing in step (2) (3) is reacted 20-240 minute (preferably 30-120 minute) under 10-35 DEG C (preferably 15-28 DEG C); With the centrifugal desalination of Zeba desalting column, remove unnecessary uncrosslinked Traut ' sreagent and Sulfo-SMCC, desalination buffer components is reaction buffer B.
(5) by obtain in step (4) with coupling agent Traut ' sreagent coupling after CD 3-resisting monoclonal antibody and with coupling agent Sulfo-SMCC coupling after anti-HER 2 monoclonal antibody, after the mol ratio of 1:2-2:1 (preferably the equimolar ratio of about 1:1) mixing, react under 10-42 DEG C (preferably 15-37 DEG C) 10-90 minute (preferably 20-60 minute).In another preference, the composition of the reaction buffer A described in step (1) is: 150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH8.0.
In another preference, the composition of the reaction buffer B described in step (2) is: 150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH7.2.
In another preference, Traut ' the s reagents described in step (3) and CD 3-resisting monoclonal antibody mol ratio are 5:1-10:1; Sulfo-SMCC and anti-HER 2 monoclonal antibody mol ratio are 4:1-20:1.
In another preference, the monoclonal antibody described in step (4) and coupling agent reaction condition are react 60 minutes at 15-28 DEG C; Desalination buffer components is reaction buffer B(150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH7.2).
In another preference, with the CD 3-resisting monoclonal antibody after coupling agent Traut ' s reagent coupling with after carrying out coupling after mixing with the anti-HER 2 monoclonal antibody equimolar ratio after coupling agent Sulfo-SMCC coupling in step (5), bi-specific antibody Conjugate ratio can be made up to 40%.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows bi-specific antibody coupling CIK cell treatment process chart.
Fig. 2 shows bi-specific antibody coupling technology schematic diagram.
Fig. 3 shows bi-specific antibody coupling CIK cell structural representation.
Fig. 4 shows coupling agent and antibody response mol ratio to the impact of coupling efficiency.Wherein: M represents molecular weight standard (Marker); 1 represents CD 3-resisting monoclonal antibody; 2 represent anti-HER 2 monoclonal antibody; 3 representatives, Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=20:1; 4 representatives, Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=40:1; 5 representatives, Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=60:1; 6 represent other antibody coupling Comparative result.
Fig. 5 shows the impact of reaction temperature, response time, reaction mol ratio antagonist coupling efficiency.Wherein, M:Marker; 1: CD 3-resisting monoclonal antibody; 2: anti-HER 2 monoclonal antibody; CD 3-resisting monoclonal antibody 37 DEG C after 3: coupling Traut ' s reagent hatches 30 minutes; 4:Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=20:1 reacts 30 minutes at 37 DEG C; 5:Traut ' s reagent: CD 3-resisting monoclonal antibody=5:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=4:1 reacts 30 minutes at 37 DEG C; CD 3-resisting monoclonal antibody after 6: coupling Traut ' s reagent 4 DEG C of overnight incubation (16 hours); 7:Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=20:1 reacts spend the night (16 hours) at 4 DEG C; 8:Traut ' s reagent: CD 3-resisting monoclonal antibody=5:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=4:1 reacts spend the night (16 hours) at 4 DEG C.
Fig. 6 shows antibody coupling efficiency electrophoresis detection collection of illustrative plates.Wherein, M:Marker; 1: CD 3-resisting monoclonal antibody; 2: anti-HER 2 monoclonal antibody; CD 3-resisting monoclonal antibody after 3: coupling Traut ' s reagent; 4: the anti-HER 2 monoclonal antibody after coupling Sulfo-SMCC; 5:Traut ' s reagent: CD 3-resisting monoclonal antibody=10:1 × Sulfo-SMCC: anti-HER 2 monoclonal antibody=20:1 at room temperature reacts 30 minutes.
Fig. 7 shows bi-specific antibody final handicraft product electrophoresis detection collection of illustrative plates.Wherein, M:Marker; 1: CD 3-resisting monoclonal antibody; 2: anti-HER 2 monoclonal antibody; 3: AntiCD3 McAb × anti-HER2 bi-specific antibody.
Fig. 8 shows the specific detection of bi-specific antibody to SK-BR-3 and CIK.Wherein, PBS:PBS processed group; OKT3:125ng CD 3-resisting monoclonal antibody/10
6cIK cell processed group; Herceptin:125ng anti-HER 2 monoclonal antibody/10
6cIK cell processed group; OKT3+Herceptin:(125ng CD 3-resisting monoclonal antibody+125ng anti-HER 2 monoclonal antibody)/10
6cIK cell processed group; BsAb:50ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell processed group.
Fig. 9 shows the detection case of bi-specific antibody and CIK cell coupling saturation.
Figure 10 shows the detection case of CIK surface anti-CD49d McAb.
Figure 11 shows the detection case of CIK surface anti-HER2 monoclonal antibody.
Figure 12 shows the ratio of the anti-CIK cell of bispecific and tumor cell to the impact of fragmentation effect.
Figure 13 shows the impact of bi-specific antibody dosage on tumor-killing effect.
Under Figure 14 shows optimal conditions, bi-specific antibody kills tumor activity detection.
Figure 15 shows bi-specific antibody and contrasts the killing activity of SK-BR-3 and MCF-7.
Detailed description of the invention
The present inventor, through extensive and deep research, by improving coupling process, provides a kind of method preparing bi-specific antibody that coupling efficiency is high, method is easy first.With bi-specific antibody prepared by the inventive method, not only Conjugate ratio very high (up to about 40%), and remain the respective activity of first antibody and second antibody well.In addition, what bi-specific antibody of the present invention can significantly improve CIK kills tumor activity, and increase rate up to 2-6 doubly or higher.Complete the present invention on this basis.
Particularly, the present inventor with medical monoclonal antibody Muromondb-CD3 (CD 3-resisting monoclonal antibody medicine) and Trastuzumab (anti-HER 2 monoclonal antibody medicine) for raw material, by steps such as antibody and coupling agent coupling, desalination, antibody and antibody couplings, preparation has the high-coupling-rate AntiCD3 McAb × anti-HER2 bi-specific antibody strengthening CIK anti-tumor activity.Test shows, AntiCD3 McAb × anti-HER2 bi-specific antibody Conjugate ratio up to 40%, the prior art level far above about 10%; In addition, after AntiCD3 McAb × anti-HER2 bi-specific antibody and CIK are hatched coupling, CIK can be killed tumor activity and significantly improve about 6 times.
Term
As used herein, term " bi-specific antibody ", " bifunctional antibody conjugate " are used interchangeably, refer to the conjugate formed by coupling arm by first antibody and second antibody, this conjugate remains the activity of respective antibody, therefore has difunctional and bispecific.In the present invention, the antibody that first antibody and second antibody are normally different.Such as, first antibody is the antibody of specific binding immunocyte, and second antibody is the antibody of specific for cancer mark or tumor cell.
Antibody
In the present invention, " antibody " refers to have specific polyclonal antibody and monoclonal antibody to albumen of the present invention, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into albumen of the present invention or its fragment respectively.Antibody of the present invention can be prepared by the various technology that those skilled in that art are known.
In the present invention, not only comprise complete monoclonal or polyclonal antibody, but also comprise and there is immunocompetent antibody fragment, as Fab ' or (Fab)
2fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A; Or chimeric antibody.
HER2
ErbB-2 (English: human epidermal growth factor receptor2, is abbreviated as HER2, is also called that Neu, ErbB-2, CD340(break up group 340) or p185) be a kind of protein by ERBB2 gene code.HER2 is one of member in EGF-R ELISA (EGFR/ErbB) family.
The breast carcinoma of nearly 20-30% has the phenomenon that HER-2 excessively shows.HER-2 is a kind of accepter on cell membrane, has high specificity with human epidermal growth factor EGF.After EGF and HER-2 combines, the dimer of HER-2 can be caused, and then cause autophosphorylation and message transmission in transfer sell, finally maintain normal Growth of Cells and division.When HER-2 excessively shows, cell can cause abnormal quick growth because of overstimulation, finally causes cancer to occur.
First antibody
First antibody used in the present invention is not particularly limited, and can be that any specific binding is in the antibody of immunocyte (as CIK) or any antibody identifying immunocyte surface marker.
In the present invention, typical immunocyte comprises (but being not limited to): cytokine induced kill cell (cytokine induced killer, CIK cell), tumor infiltrating lymphocyte (tumor infiltratinglymphocyte, til cell), cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL), dendritic cell (dendritic cell, DC) or its combination.Wherein, particularly preferred immunocyte is CIK cell.
In the present invention, the preferred first antibody of a class comprises (but being not limited to): the antibody of AntiCD3 McAb, anti-CTLA 4 or its combination.
In the present invention, the preferred first antibody of a class is that specific binding is in the antibody (antibody as AntiCD3 McAb) of CIK cell.
Second antibody
Second antibody used in the present invention is not particularly limited, and can be all specific bindings in the antibody of tumor markers or tumor cell.
The preferred second antibody of one class comprises (but being not limited to): the antibody of anti-HER2, the antibody of anti-EGFR or its combination.
Coupling arm and coupling method
Coupling arm used in the present invention is not particularly limited, and can be all groups that first antibody and second antibody can be linked together.In the present invention ,-the NH of usual coupling arm and the first and second antibody constant regions (being especially positioned near constant region end or end)
2connect, thus form the conjugate that structure is " first antibody-coupling arm-second antibody ".Usually ,-the NH on antibody
2be converted into-NH-, side is connected with antibody, and side is connected with coupling molecule.In the present invention, the described-NH derived from antibody
2the imido grpup of group is regarded as the part being still antibody usually, certainly, also can be considered a part for coupling arm.
Coupling arm is formed by coupling agent usually.But the coupling efficiency of different coupling agents is different, and the impact of antagonist is also not quite similar.
In the present invention, representational coupling arm comprises (but being not limited to): the coupling arm formed by TrautShi reagent (Traut ' sreagent) and Sulfo-SMCC.
Based on different coupling agents, diverse ways can be adopted.In the present invention, a kind of preferred method adopts TrautShi reagent and Sulfo-SMCC to carry out coupling.
A kind of typical coupling method as shown in Figure 2.A kind of preparation method of preferred AntiCD3 McAb × anti-HER2 bi-specific antibody comprises the following steps:
(1) dissolving of antibody: commercialization CD 3-resisting monoclonal antibody is dissolved in reaction buffer A(150mMNaCl, 2mM EDTA ˙ Na
2, 100mM phosphate buffer, pH8.0); Anti-medical HER2 monoclonal antibody is dissolved in reaction buffer B(150mM NaCl, 2mM EDTA ˙ Na
2, 100mM phosphate buffer, pH7.2).
(2) dissolving of coupling agent: take rapidly Traut ' s reagent and Sulfo-SMCC and be dissolved in respectively in ultra-pure water, being prepared into mother solution.
(3) coupling of CD 3-resisting monoclonal antibody and Traut ' s reagent: Traut ' the sreagent mother solution prepared is mixed homogeneously with the ratio of mol ratio 5:1 ~ 10:1 with CD 3-resisting monoclonal antibody; The mixture of mix homogeneously is placed on isothermal reaction shaking table, reacts 60 minutes under maintaining the temperature at 15 ~ 28 DEG C of conditions.
(4) coupling of anti-HER 2 monoclonal antibody and Sulfo-SMCC: the Sulfo-SMCC mother solution prepared is mixed homogeneously with the ratio of mol ratio 4:1 ~ 20:1 with anti-HER 2 monoclonal antibody; The mixture of mix homogeneously is placed on isothermal reaction shaking table, reacts 60 minutes under maintaining the temperature at 15 ~ 28 DEG C of conditions.
(5) desalting column desalination: Zeba desalting column is used in advance reaction buffer B(150mM NaCl, 2mMEDTA ˙ Na
2, 100mM phosphate buffer, pH7.2) and balance, according to desalting column operation instruction by above-mentioned steps (3), the CD 3-resisting monoclonal antibody of having reacted in (4) and anti-HER 2 monoclonal antibody are splined on desalting column, measure antibody protein concentration after centrifugal eluting.
The coupling of CD 3-resisting monoclonal antibody and anti-HER 2 monoclonal antibody: the CD 3-resisting monoclonal antibody of concentration known in step (5) is reacted 30 minutes after mixing with anti-HER 2 monoclonal antibody equimolar ratio under 28 ~ 37 DEG C of conditions, or react spend the night (16 hours) under 4 DEG C of conditions, finally obtain object product AntiCD3 McAb × anti-HER2 bi-specific antibody.
Bifunctional protein and preparation thereof
In the present invention, " bifunctional antibody conjugate ", " conjugate of the present invention ", " bi-specific antibody of the present invention ", " bispecific conjugate ", " BsAb " are used interchangeably, refer to that there is structure described in formula Ia or Ib, containing the bifunctional antibody conjugate of specificity for the second antibody of the first antibody of immunocyte, coupling arm and specific for cancer mark.A representational example is AntiCD3 McAb × anti-HER2 bi-specific antibody.The polymer that conjugate of the present invention can be conjugate (i.e. dimer) or be formed further by conjugate.In addition, should be understood that described term also comprises active fragment and the derivant of bifunctional antibody conjugate.
A kind of typical conjugate is AntiCD3 McAb × anti-HER2 bi-specific antibody, and its structural representation as shown in Figure 3.This conjugate is the synthetic antibody containing AntiCD3 McAb and anti-HER2 two species-specific antigen binding site, can at HER2 positive cell and tumor-killing cell cytokine induced kill cell (Cytokine-inducedkiller, CIK) bridge is erected between, excite the immunoreation with guidance quality, have broad application prospects in the immunization therapy of tumor.
Pharmaceutical composition and application process
Present invention also offers a kind of compositions, it contains the bifunctional antibody conjugate of the present invention of effective dose, and pharmaceutically acceptable carrier.Usually, bifunctional antibody conjugate of the present invention can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.
As used herein, term " effective dose " or " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts, as 0.000001-90wt%; Preferably 0.1-50wt%; Better, 5-40wt%.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.
Pharmaceutical composition of the present invention contains the bifunctional antibody conjugate of the present invention of safe and effective amount and pharmaceutically acceptable carrier.This kind of carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into solution form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described pharmaceutical composition should aseptically manufacture.The dosage of active component is treatment effective dose.
The effective dose of bifunctional antibody conjugate of the present invention can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: pharmacokinetic parameter such as bioavailability, metabolism, the half-life etc. of described bifunctional antibody conjugate; The order of severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.Usually, when bifunctional antibody conjugate of the present invention gives with the dosage of about 0.0001mg-50mg/kg the weight of animals (preferably 0.001mg-10mg/kg the weight of animals) every day, gratifying effect can be obtained.Such as, by an urgent demand for the treatment of situation, the dosage that several times separate can be given every day, or dosage is reduced pari passu.
Conjugate of the present invention is preferably utilized to carry out a method for cell therapy, as shown in Figure 1.Hatch by conjugate of the present invention (as AntiCD3 McAb × anti-HER2 bi-specific antibody) and immunocyte (as CIK), thus form " conjugate-immunocyte " complex (as BsAb-CIK complex).Then, " conjugate-immunocyte " complex can be made further the dosage form of applicable administration, and carry out administration.
In addition, in the present invention, the mixed proportion of conjugate of the present invention and immunocyte (as CIK) is generally 20-100ng:0.5 × 10
6~ 5 × 10
7cell is preferably 20-100ng:0.5 × 10
6~ 5 × 10
6cell, as 50ng:1 × 10
6cell.
A kind of method of representational AntiCD3 McAb × anti-HER2 bi-specific antibody coupling CIK cell, comprises the following steps: anti-OKT3 × anti-HER2 bi-specific antibody coupling CIK cell: CIK cell concentration is adjusted to 1 × 10
6individual/mL, according to 50ng bi-specific antibody/10
6individual CIK cell ratio adds anti-OKT3 × anti-HER2 bi-specific antibody, hatches after 30 minutes with PBS cleaning 1 ~ 2 time under 4 DEG C of conditions under hatching 1 hour or room temperature condition.
The major advantage of bifunctional antibody conjugate of the present invention comprises:
1) simple, the strong operability of preparation technology;
2) synthesize by present invention process the AntiCD3 McAb × anti-HER2 bi-specific antibody Conjugate ratio obtained and reach 40%, far above at present only about 10% prior art level;
3), after bi-specific antibody of the present invention and CIK being hatched coupling, CIK can be killed tumor activity and improve 2-6 doubly.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number are percentage by weight and parts by weight.
Embodiment 1
The preparation of AntiCD3 McAb × anti-HER2 bi-specific antibody
Experimental procedure:
Get mouse-anti people CD3 monoclonal antibody 132 μ g and be dissolved in reaction buffer A(150mM NaCl, 2mMEDTANa
2100mM phosphate buffer, pH8.0), take the reaction buffer A that Traut ' s reagent1.2mg is dissolved in 120 μ L, be mixed with 10mg/mL mother solution, during use with dilution 50 times to 0.2mg/mL, Traut ' s reagent and CD 3-resisting monoclonal antibody are mixed with mol ratio 10:1 ratio and hatch 1 hour at ambient temperature;
Get Mus anti-HER 2 monoclonal antibody 30 μ g and be dissolved in reaction buffer B(150mM NaCl, 2mMEDTANa
2100mM phosphate buffer, pH7.2), take the reaction buffer B that Sulfo-SMCC1.5mg is dissolved in 300 μ L, be mixed with mother solution, dilute 10 times during use to 0.5mg/mL, Sulfo-SMCC and anti-HER 2 monoclonal antibody are mixed with mol ratio 20:1,40:1,60:1 ratio and hatches 1 hour at ambient temperature;
Zeba desalting column is used in advance reaction buffer B(150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH7.2) and balance, according to desalting column operation instruction, the CD 3-resisting monoclonal antibody of having reacted in above-mentioned steps and anti-HER 2 monoclonal antibody are splined on desalting column, after centrifugal eluting, measure antibody protein concentration.
Under 28 DEG C of conditions, react 30 minutes after the CD 3-resisting monoclonal antibody of the concentration known above step obtained mixes with anti-HER 2 monoclonal antibody equimolar ratio, finally obtain object product AntiCD3 McAb × anti-HER2 bi-specific antibody.
The concentration of antibody adopts IgG parameter in Nanodrop2000 instrument to be measured fast and accurately, and substantially reduce antibody concentration minute, its accuracy and reliability are calibrated through BCA determination of protein concentration method.
Experimental result:
As shown in Figure 4, Traut ' s reagent and CD 3-resisting monoclonal antibody are with mol ratio 10:1, Sulfo-SMCC and anti-HER 2 monoclonal antibody with under mol ratio 20:1 condition, and antibody coupling efficiency reaches about 50%.
Embodiment 2
The preparation of AntiCD3 McAb × anti-HER2 bi-specific antibody
Different from embodiment 1, concrete steps are as follows:
Get mouse-anti people CD3 monoclonal antibody OKT3 antibody 133 μ g and be dissolved in reaction buffer A(150mM NaCl, 2mM EDTANa
2100mM phosphate buffer, pH8.0), take the reaction buffer A that Traut ' s reagent1.3mg is dissolved in 130 μ L, be mixed with 10mg/mL mother solution, dilute 50 times during use to 0.2mg/mL, Traut ' s reagent and CD 3-resisting monoclonal antibody are blended under room temperature bar adds with mol ratio 5:1 or 10:1 ratio and hatch 1 hour;
Get Mus anti-HER 2 monoclonal antibody 31 μ g and be dissolved in reaction buffer B(150mM NaCl, 2mMEDTANa
2100mM phosphate buffer, pH7.2), take the reaction buffer B that Sulfo-SMCC1.0mg is dissolved in 100 μ L, be mixed with mother solution, dilute 20 times during use to 0.5mg/mL, Sulfo-SMCC and anti-HER 2 monoclonal antibody are blended under room temperature bar adds with mol ratio 4:1 or 20:1 ratio and hatch 1 hour;
Zeba desalting column is used in advance reaction buffer B(150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH7.2) and balance, according to operation instruction, the CD 3-resisting monoclonal antibody of having reacted in above-mentioned steps and anti-HER 2 monoclonal antibody are splined on desalting column, after centrifugal eluting, measure antibody protein concentration.
React under 37 DEG C of conditions 30 minutes or react spend the night (16 hours) under 4 DEG C of conditions after the CD 3-resisting monoclonal antibody of the concentration known above step obtained mixes with anti-HER 2 monoclonal antibody equimolar ratio, finally obtain object product AntiCD3 McAb × anti-HER2 bi-specific antibody.
Experimental result:
As shown in Figure 5, react 30 minutes under 37 DEG C of conditions or react the coupling efficiency that spend the night (16 hours) do not affect antibody under 4 DEG C of conditions, the Conjugate ratio of antibody after coupling agent and antibody molar ratio being reduced, can be reduced.
Embodiment 3
The detection of AntiCD3 McAb and anti-HER 2 monoclonal antibody Conjugate ratio
Specific experiment step:
Get mouse-anti people CD3 monoclonal antibody OKT3 antibody 600 μ g and be dissolved in reaction buffer A(150mM NaCl, 2mM EDTANa
2100mM phosphate buffer, pH8.0), take the reaction buffer A that Traut ' s reagent1.2mg is dissolved in 120 μ L, be mixed with mother solution, dilute 100 times during use to 0.1mg/mL, Traut ' s reagent and CD 3-resisting monoclonal antibody are blended under room temperature bar adds with mol ratio 10:1 ratio and hatch 1 hour;
Get Mus anti-HER 2 monoclonal antibody 600 μ g and be dissolved in reaction buffer B(150mM NaCl, 2mMEDTANa
2100mM phosphate buffer, pH7.2), take the reaction buffer B that Sulfo-SMCC0.3mg is dissolved in 300 μ L, be mixed with mother solution, dilute 10 times during use to 0.1mg/mL, Sulfo-SMCC and anti-HER 2 monoclonal antibody are blended under room temperature bar adds with mol ratio 20:1 ratio and hatch 1 hour;
Zeba desalting column is used in advance reaction buffer B(150mM NaCl, 2mM EDTANa
2, 100mM phosphate buffer, pH7.2) and balance, according to operation instruction, the CD 3-resisting monoclonal antibody of having reacted in above-mentioned steps and anti-HER 2 monoclonal antibody are splined on desalting column, after centrifugal eluting, measure antibody protein concentration.
React 30 minutes at ambient temperature after the CD 3-resisting monoclonal antibody of the concentration known above step obtained mixes with anti-HER 2 monoclonal antibody equimolar ratio, finally obtain object product AntiCD3 McAb × anti-HER2 bi-specific antibody.
Antibody (AntiCD3 McAb and anti-HER 2 monoclonal antibody) before getting coupling, to mix with 5 μ L sample-loading buffers with antibody after coupling agent coupling (CD 3-resisting monoclonal antibody of coupling Traut ' s reagent and the anti-HER 2 monoclonal antibody of coupling Sulfo-SMCC) and each 15 μ L of monoclonal antibody mutual coupling afterproduct, 70 DEG C of processing sample 10min or 100 DEG C processing sample 5-10min, are splined on NuPAGE Tris-Acetate Mini Gels and carry out irreducibility SDS-PAGE electrophoretic analysis.Electrophoresis 60-80min under 200V voltage.Get glue coomassie brilliant blue staining after electrophoresis terminates, take pictures after destaining solution decolouring, BIORAD gel imaging software analysis monomer, dimer and the general proportions contained by polymer.
Experimental result
As shown in Figure 6, show anti-monoclonal antibody with coupling agent coupling after self-crosslinking degree lower, the bi-specific antibody of display acquisition 30% Conjugate ratio after crosslinked between antibody.
As shown in Figure 7, the final AntiCD3 McAb × anti-HER2 bi-specific antibody Conjugate ratio after optimizing reaches about 40%, and wherein monomer accounts for 40%, and dimer accounts for 40%, and polymer accounts for 20%, higher than reporting level at present.
Embodiment 4
AntiCD3 McAb × anti-HER2 bi-specific antibody detects CIK cell and HER2 positive tumor cell specific binding
Experiment concrete steps are as follows:
CIK cell concentration is adjusted to 2 × 10
6individual/mL, respectively get 1mL and be divided into 5 groups, divide and add PBS, 250ng CD 3-resisting monoclonal antibody, 250ng anti-HER 2 monoclonal antibody, 250ng CD 3-resisting monoclonal antibody+250ng anti-HER 2 monoclonal antibody, 100ng AntiCD3 McAb × anti-HER2 bi-specific antibody, 30 minutes are hatched under room temperature bar adds, after cleaning 2 times with complete medium, every component gets 100 μ L(2 × 10
5individual cell) add in 96 porocyte culture plates.Adjustment breast carcinoma HER2 positive cell SK-BR-3 concentration is to 2 × 10
5individual/mL, adds 50 μ L(1 × 10 in every hole
4) make E:T reach 20:1, be placed in cell culture incubator and cultivate microscopy after 12 hours.
Experimental result:
As shown in Figure 8, CIK cell targeting can be positioned tumor cell by AntiCD3 McAb × anti-HER2 bi-specific antibody effectively.
Embodiment 5
The detection of AntiCD3 McAb × anti-HER2 bi-specific antibody and CIK cell coupling saturation
Experiment concrete steps are as follows:
CIK cell concentration is adjusted to 1 × 10
6, be divided into 5 parts, add PBS, 5ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10 respectively
6cIK cell, 50ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell, 100ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell, 500ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell.Be placed in 4 DEG C hatch 1 hour after, 2 times are cleaned with the PBS containing 1%BSA, clean 2 times with PBS after hatching 30 minutes under adding sheep anti-mouse igg streaming antibody 4 DEG C of lucifuge conditions of PE labelling, adjust last volume greatly about 200 μ L, be splined on flow cytometer and detect.
Experimental result:
As shown in Figure 9,50ng bi-specific antibody and 10
6individual CIK cell can close to saturated after giving, prompting 50ng bi-specific antibody/10
6individual CIK cell can reduce to the full extent in conjunction with CIK cell on drug dose basis, plays anti-tumor activity.
Embodiment 6
The detection of AntiCD3 McAb × anti-HER2 bi-specific antibody and CIK cell combination rate
Specific experiment step is as follows:
1. the detection of the anti-CD monoclonal antibody of cell surface: CIK cell concentration is adjusted to 1 × 10
6, be divided into 3 parts, add PBS, 125ng CD 3-resisting monoclonal antibody/10 respectively
6cIK cell, 50ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell.Be placed in 4 DEG C hatch 1 hour after, clean 2 times with containing the PBS of 1%BSA, add the sheep anti-mouse igg 2a of streaming antibody PE labelling, clean 2 times after 4 DEG C of lucifuges hatch 30 minutes with PBS, adjustment volume, greatly about 200 μ L, is splined on flow cytometer and detects.
2. the detection of cell surface anti-HER 2 monoclonal antibody: CIK cell concentration is adjusted to 1 × 10
6, be divided into 3 parts, add PBS, 125ng CD 3-resisting monoclonal antibody/10 respectively
6cIK cell, 50ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK cell.Be placed in 4 DEG C hatch 1 hour after, clean 2 times with containing the PBS of 1%BSA, clean 2 times with PBS after the sheep anti-mouse igg Isosorbide-5-Nitrae DEG C lucifuge adding streaming antibody FITC labelling hatches 30 minutes, adjustment volume, greatly about 200 μ L, is splined on flow cytometer and detects.
Experimental result:
As shown in Figure 10, bi-specific antibody and CIK cell hatch rear fluorescence signal intensity and the independent incubated cell of AntiCD3 McAb is basically identical.
As shown in figure 11, cell surface anti-HER 2 monoclonal antibody combination rate reaches about 10%, shows that part bi-specific antibody has been coupled to CIK cell surface.
Embodiment 7
Lethal effect to HER2 positive breast cancer cells SK-BR-3 after AntiCD3 McAb × anti-HER2 bi-specific antibody coupling CIK
Specific experiment step is as follows:
CIK cell concentration is adjusted to 4 × 10
6individual/mL(and corner block plaid cell number amount to 1600, each large lattice 400 cells), 8mL altogether.Every part of 2mL, according to 0,5,50,100ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6cIK adds 0 respectively, 40,400,800ng AntiCD3 McAb × anti-HER2 bi-specific antibody (is 1mg/mL according to AntiCD3 McAb × anti-HER2 bi-specific antibody initial concentration, i.e. 1000ng/ μ L, dilute antibody in proportion, add in CIK, 15 minutes are hatched under room temperature condition, with complete medium cleaning twice, each centrifugal rotational speed 400g, 5 minutes.SK-BR-3 cell concentration is adjusted to 4 × 10
5individual/mL(and corner block plaid cell number amount to 160, each large lattice 40 cells), 3.5mL altogether, in 96 porocyte culture plates, every hole adds 50 μ L.CIK and SK-BR-3 tumor cell is added in 96 porocyte culture plates according to the ratio of 5:1,10:1,20:1,30:1, requires to carry out cytotoxicity detection according to Promega test kit description.
Experimental result
As shown in figure 12, under CIK cell saturated coupling AntiCD3 McAb × anti-HER2 bi-specific antibody condition, different E:T experiments shows, when E:T reaches 20:1, the CIK of bi-specific antibody coupling kills tumor activity and reaches more than 30%, and the CIK of non-coupling bi-specific antibody kills tumor activity only reaches about 5%, bi-specific antibody makes the tumor activity that kills of CIK significantly improve, and at least improves 6 times.
As shown in figure 13, under E:T reaches 20:1 condition, 50ng bi-specific antibody/10
6cIK can make the tumor activity that kills of CIK bring up to about 40%, and no longer improves along with the raising anti-tumor activity of bi-specific antibody dosage.
As shown in figure 14, hatch CIK cell by two kinds of monoclonal antibodies of monoclonal antibody or non-coupling separately and can not improve the killing activity of CIK to tumor cell, demonstrate the effect that bi-specific antibody plays in tumor-killing process further.
Embodiment 8
AntiCD3 McAb × anti-HER2 bi-specific antibody is to the comparison of HER2 high expressed amount and the effect of low expression tumor cytotoxicity
Specific experiment step is as follows:
CIK cell concentration is adjusted to 4 × 10
6individual/mL, according to 50ng AntiCD3 McAb × anti-HER2 bi-specific antibody/10
6according to embodiment 7 step process after CIK measures and hatches, in 96 porocyte culture plates, add 100 μ L/ holes; The MCF-7 breast cancer cell concentration of the SK-BR-3 breast cancer cell of HER2 high expressed amount and the low expression of HER2 is adjusted to 4 × 10 respectively
5individual/mL, adds 50 μ L/ holes, requires to carry out cytotoxicity detection according to Promega test kit description.
Experimental result:
As shown in figure 15, the MCF-7 of the CIK cell after bi-specific antibody coupling to the low expression of HER2 has lethal effect equally, but lower than the SK-BR-3 of HER2 high expressed amount.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a bifunctional antibody conjugate, is characterized in that, described bifunctional antibody conjugate has structure described in formula Ia or formula Ib:
A-B-C (Ia), or
C-B-A (Ib)
Wherein,
A is the first antibody of specificity for immunocyte;
B is the coupling arm between A and C;
C is the second antibody of specific for cancer mark;
"-" represents the chemical bond connecting said elements.
2. bifunctional antibody conjugate as claimed in claim 1, it is characterized in that, described first antibody is selected from lower group: the antibody of AntiCD3 McAb, anti-CTLA 4 antibody or its combination; And/or
Described second antibody is selected from lower group: the antibody of anti-HER2, the antibody of anti-EGFR or its combination.
3. bifunctional antibody conjugate as claimed in claim 1, it is characterized in that, described antibody coupling matter has one or more characteristics following:
A) can simultaneously with CIK cell and these two kinds of Cell bindings of tumor cell;
B) can (6,12,24 months or longer) preserve for a long time under 4 DEG C or-20 DEG C of conditions.
4. bifunctional antibody conjugate as claimed in claim 1, it is characterized in that, described coupling arm is selected from lower group:
In formula, "-" of each structural formula both sides represents the key (chemical bond or covalent bond) be connected with second antibody with first antibody respectively;
Preferably, described bifunctional antibody conjugate structure is as follows:
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody;
And-the HN-be connected with X with Z or-NH-represents the-NH derived from antibody
2the imido grpup of group.
5. bifunctional antibody conjugate as claimed in claim 1, it is characterized in that, described first antibody is anti-cd 3 antibodies, and described second antibody is Anti-HER 2.
6. a pharmaceutical composition, is characterized in that, described compositions contains arbitrary described bifunctional antibody conjugate in claim 1-4, and pharmaceutically acceptable carrier.
7. a method for pharmaceutical compositions, is characterized in that, comprises step: mixed with pharmaceutically acceptable carrier by described bifunctional antibody conjugate arbitrary in claim 1-4, thus obtained pharmaceutical composition.
8. prepare a method for antitumor drug, it is characterized in that, comprise step:
A bifunctional antibody conjugate according to claim 1 mixes with immunocyte by (), thus form the complex of " bifunctional antibody conjugate-immunocyte ",
B described complex is made antitumor drug by ().
9. the purposes of bifunctional antibody conjugate as claimed in claim 1, is characterized in that, for the preparation of the medicine of disease therapy;
Preferably, described disease comprises tumor.
10. produce a method for bifunctional antibody conjugate according to claim 1, it is characterized in that, it comprises step:
A () provides the first intermediate shown in formula II; With the second intermediate shown in formula III
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody;
B the second intermediate shown in the first intermediate shown in formula II and formula III is carried out coupling reaction by (), thus form the bifunctional antibody conjugate shown in formula IV:
In formula,
X is first antibody A or second antibody C;
Z is first antibody A or second antibody C; Further, in X and Z, one is first antibody, and another is second antibody.
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| CN109939232A (en) * | 2017-12-21 | 2019-06-28 | 张曼 | The application of killing bladder cancer cell PUMC-91 is oriented about CD3 × B7H3 bispecific antibody |
| CN109939230A (en) * | 2017-12-21 | 2019-06-28 | 张曼 | The application of resistance to cis-platinum bladder cancer cell T24/DDP is killed about CD3 × B7H3 bispecific antibody orientation |
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