CN103214578B - Novel humanized anti-CD22 antibody - Google Patents
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Abstract
The invention relates to the field of bioengineering, and specifically relates to a novel humanized anti-CD22 antibody. The novel humanized anti-CD22 antibody takes a murine antibody RFB4 as a parental antibody and uses a CDR implantation technique to carry out humanization; and the humanized new antibody hRFB4 has affinity similar with the parental antibody, and keeps the bonding capability of the parental antibody with the CD22 antigen and the cell internalizing activity of antigen-antibody complex caused by the antibody. Compared with the parental antibody, the humanized protein sequence proportion in the humanized hRFB4 antibody reaches over 90%, so that the immunogenicity is greatly reduced and the occurrence of HAMA reaction is avoided.
Description
Technical field:
The present invention relates to a kind of novel humanization anti-CD22 antibody, belong to technical field of bioengineering.
Background technology:
Non-Hodgkin lymphoma (Non-Hodgkin lymphoma, NHL) is one group and originates from lymphoglandula and other adenoid malignant tumours, is lymphadenomatous one large type (except Hodgkin lymphoma).Main clinical manifestation can be summarized as follows: 1, superficial lymph knot enlargement or formation tubercle lump; 2, deep lymph node lump in body; 3, hyperplasia and the lump of knot perilymph tissue; 4, constitutional symptom, comprises heating, night sweat, weak and lose weight.To increase the second fast cancer in this disease of the U.S., with the speed increase of newly-increased approximately 55,000 examples every year.In recent years, China's malignant lymphoma sickness rate rises and accelerates, and has entered ten large tumour ranks occurred frequently, and the sickness rate of China NHL has rapidly increased to 7,/10 ten thousand from 2,/10 ten thousand of eighties of last century.
Systemic lupus erythematous (systemic lupus erythematosus, SLE) be a kind of diffusivity, general autoimmune disease, mainly involve mucocutaneous, skeletal muscle, kidney and central nervous system, can also involve multiple organs and the systems such as lung, heart, blood, have various clinical symptoms simultaneously; In serum, autoantibody and crucial immunological abnormality can be detected.Whole world SLE morbidity is 48,/10 ten thousand-1,17/,100,000, and sickness rate reaches 1.5/10 ten thousand-7.6/10 ten thousand; The multiple women at reproduction age who is born in 20～40 years old, the ratio of men and women's sickness rate is about 1: 7～and 1: 10, various countries crowd's physical and mental health in its high morbidity, high case fatality rate serious threat.
In the past few decades, the primary treatment method of non-Hodgkin lymphoma and systemic lupus erythematous is radiation and chemotherapy, but being developed as us new treatment plan being provided of monoclonal antibody drug can make patient effectively avoid dependence and the toxic side effect to radiotherapy chemotherapy.U.S. FDA approval anti-CD20 antibodies Mabthera (Rituximab) listing is a historical milestone.Although Mabthera to NHL effectively and toxicity can control, but not every patient has therapeutic response to it, each has the rear portion of patient of therapeutic response to recur in fact, but Mabthera encourages investigators to remove to find new target spot (Lu Pingping etc., Chinese experimental and liquid are learned magazine, 2006; 14 (6): 1258-1261), the phraseology of these target spots is different from CD20 with physilogical characteristics, but may more effectively maybe can treat the B cell lymphoma patient that Mabthera is invalid than Mabthera.
CD22 antigen is in numerous lymphocytic cell surface target spots, approximately the B cell malignancies of 60-80% is expressed CD22, becomes thus potential target (Cesano, the A. of passive immunization therapy, Gayko, U. (2003) Semin.Oncol.30:253-257).
CD22 antigen, claims again the immunoglobulin-like agglutinant 2 (SIGLEC2) of sialic acid combination, is a kind of II type transmembrane glycoprotein of B cell, has α, two kinds of different hypotypes of β, and molecular weight is about 140KD left and right.The extracellular region of CD22 is made up of 7 immunoglobulin domains, and its N end structure territory is V-immunoglobulin domains, is responsible for and α 2, and 6Sia part combines; Say it is also a member of immunoglobulin superfamily from functional perspective,, there is close relationship on the restricted surface that is expressed in mature B cell and most of B cell NHL with development, differentiation and the function of B cell.
The biological function of CD22 molecule is by conditioning signal conduction path, and ligand binding domains regulates the inhibition of B cell signaling, participates in B cell directly and reaches anti-knurl effect to aspects such as going back to the nest of marrow.Its feature is to weaken the interaction of immunogenicity and the reinfocing effect factor.Through the painstaking efforts of nearly 20 years, anti-CD22 antibody treatment NHL made great progress.
At present, have in the antibody drug take CD22 antigen as target spot in clinical study stage: the humanized antibody of Immunomedics company---epratuzumab (Epratuzumab); The Inotuzumab ozogamicin of Hui Shi is and the humanization monoclonal antibody of chemical toxicant coupling; The BL22 of NIH is the immunotoxin that has merged PE38 on the basis of murine antibody; And the SM03 chimeric antibody of the auspicious medicine company of Shenzhen dragon etc.
Epratuzumab is the IgG1 class of Humanized monoclonal antibodies LL2, and directly target CD22 antigen, can mediate corresponding protein phosphorylation in endochylema by CD22, promotes Raji clone apoptosis, suppresses its differentiation, thereby reaches the lymphadenomatous object for the treatment of Burkitt ' s.Inotuzumab Ozogamicin (CMC-544) is a kind of antibody target chemotherapeutics, formed by the covalently bound tumour microbiotic of humanization anti-CD22 antibody calicheamicin (Enediyne high efficiency anti-tumor microbiotic), it is a kind of potent cytotoxic agent, can effectively resist CD22B Cell Non-Hodgkin's, main adverse reaction is reversibility thrombopenia.BL22 (RFB4 (dsFv)-PE38) is recombinant immunotoxin.BL22 is that RFB4 passes through the disulfide linkage of cysteine residues and the connector of variable region heavy chain, then merges and become RFB4 (dsFv)-PE38 with PE38.BL22 is the immunotoxin that produces high complete reaction rate in first hairy cell leukemia (hairy cell leukemia, HCL) patient who suppresses at purine homologue.Research patients' HCL such as Kreitman the clinical I phase experimental results show that BL22 can tolerate with highly effective.Although HCL produces complete reaction to BL22, BL22 is lower to the low patient's curative effect of CD22 antigen presentation.Investigator wishes to undergo mutation to increase antibody affinity and curative effect at complement determining area (CDR), but not success.SM03 chimeric antibody is to clone respectively mouse-anti people CD22 monoclonal antibody light/heavy chain variable region gene VL/VH, builds restructuring SM03 chimeric antibody.SM03 chimeric antibody is specific to B cell CD 22 antigen, and malignant lymphatic oncocyte is had to growth inhibitory effect, for the lymphadenomatous clinical treatment of malignant B cell and application are laid a good foundation.
Summary of the invention:
The present invention on the basis of existing technology, develops the anti-CD22 monoclonal antibody of a kind of novel humanization.Take mouse source RFB4 as parental antibody, adopt CDR implantation technique to carry out humanization to it, parental antibody CDR district is transplanted in human IgG1, and the protein structure model of the CD22 antibody of appliance computer macromole simulation technique after to humanization is analyzed, final definite 6 amino acid sites that suddenly change, as shown in italics part in sequence, by the method for synthetic degenerate primer, build the humanized antibody hRFB4 phage antibody library that comprises 64 kinds of polymorphisms.This phage antibody library is shown and filtered out 5 kinds of antibody dna sequences that avidity is higher, and translation obtains corresponding aminoacid sequence according to nucleotide sequence.The gene order of relatively finding these 5 humanization hRFB4 antibody by sequential analysis there are differences at 4 different amino acid sites respectively, but they can be combined with CD22 antigen, show higher affinity of antibody, there is similar biological function.
By 5 humanized CD22 antibody---hRFB4, called after YQ22-1 to YQ22-5 respectively, the aminoacid sequence of the variable region of heavy chain of five antibody is SEQ ID NO:1, the aminoacid sequence of variable region of light chain is respectively as SEQ ID NO:2,3,4, shown in 5,6, specific as follows:
YQ22-1：
V
H：EVQLVESGGGLVQPGGSLRLSCAASGFTFS
WVRQAPGKGLEWVS
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
WGQGTLVTVSS[SEQ?ID?NO：1]
V
L：DIQMTQSPSSLSASVGDRVTINC
WYQQKPGKAPKLLIY
GVPSRFSGSGSGTDFSLTISSLQPEDFATYFC
FGQGT[SEQ?ID?NO：2]
YQ22-2：
V
H：[SEQ?ID?NO：1]
V
L：DIQMTQSPSSLSASVGDRVTITC
WYQQKPGKAPKLLIY
GVPSRFSGSGSGTDYTLTISSLQPEDFATYFC
FGQGT[SEQ?ID?NO：3]
YQ22-3：
V
H：[SEQ?ID?NO：1]
V
L：DIQMTQSPSSLSASVGDRVTITC
WYQQKPGKAPKLLIY
GVPSRFSGSGSGTDFSLTISSLQPEDFATYFC
FGQGT[SEQ?ID?NO：4]
YQ22-4：
V
H：[SEQ?ID?NO：1]
V
L：DIQMTQSPSSLSASVGDRVTITC
WYQQKPGKAPKLLIY
GVPSRFSGSGSGTDFSLTISSLQPEDFATYYC
FGQGT[SEQ?ID?NO：5]
YQ22-5：
V
H：[SEQ?ID?NO：1]
V
L：DIQMTQSPSSLSASVGDRVTISC
WYQQKPGKAPKLLIY
GVPSRFSGSGSGTDYTLTISSLQPEDFATYFC
FGQGT[SEQ?ID?NO：6]
The object of the present invention is to provide above-mentioned novel humanization anti-CD22 antibody, the DNA sequence dna that comprises encoding antibody molecule, carrier, host cell, preparation method and described antibody molecule are treated by the purposes in the cell-mediated disease of expression CD22 in preparation.
Humanized antibody hRFB4 of the present invention, has the avidity similar to mouse source parental antibody RFB4, can specific combination CD22 antigen, and kept causing the biological characteristics of cell-surface antigens antibody complex internalization.Meanwhile, the antibody after humanization, people's heterologous protein sequence ratio reaches more than 90%, has greatly reduced the immunogenicity that murine antibody causes, has avoided HAMA reaction.
Technical scheme of the present invention is as follows:
The monoclonal antibody that the invention provides a kind of specific binding people CD22, is made up of variable region and constant region, the framework region in the CDR that variable region comprises mouse source and people source, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ ID NO:1; The aminoacid sequence of variable region of light chain is respectively as shown in SEQ ID NO:2,3,4,5,6;
Described monoclonal antibody is the humanized antibody that CDR transplants;
The constant region of described monoclonal antibody is any one of human IgG1, IgG2, IgG3 or IgG4; Be preferably human IgG1;
The invention provides the carrier of the polynucleotide sequence that comprises encoding said antibody; Described carrier is pCDNA3.1;
The invention provides the host cell of the above-mentioned carrier of a kind of transfection;
The invention provides the preparation method of described antibody, comprise the DNA expression vector transfection host cell that uses the described monoclonal antibody of coding, obtain the cell culture supernatant that contains described antibody, and prepare target protein through Protien A column purification;
The invention provides described antibody for the preparation for the treatment of by the purposes in the cell-mediated disease of expression CD22; Described disease is malignant lymphoma and autoimmune disorder, specifically refers to non-Hodgkin lymphoma and systemic lupus erythematous.
Accompanying drawing explanation:
Fig. 1 is humanization hRFB4 antibody and mouse source RFB4 antibody weight chain amino acid comparison diagram.
Fig. 2 is humanization hRFB4 antibody Space structure simulation figure.
Fig. 3 is hRFB4 antibody weight chain expression vector plasmid map.
Fig. 4 is hRFB4 antibody SDS-PAGE electrophorogram.
Fig. 5 is hRFB4 antibody concentration mensuration figure.
Fig. 6 is that the affinity of antibody based on ELISA method detects.
Fig. 7 is the combination test of hRFB4 antibody cell strain surface C different from three kinds D22 antigen.
Fig. 8 is the CD22 antigen internalization effect that hRFB4 antibody causes.
Specific implementation method:
Embodiment of the present invention illustrate by the following example.But, should be appreciated that embodiment of the present invention are not limited to the specific detail of these embodiment, because for the person of ordinary skill of the art, other variation is known, or is apparent according to direct disclosed content and appended claims.Therefore, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.The reference of quoting is herein incorporated to herein in full by reference with it.
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
The humanization of embodiment 1, RFB4 antibody
RFB4 is light, heavy chain gene is from NCBI, and nucleic acid encoding is respectively: CS110728, CS110730.DNA sequence dna is translated into after aminoacid sequence, according to Kabat rule, simultaneously with reference to pertinent literature and patent, analyzed framework region (FR) and complementary determining region (CDR) of RFB4 variable region gene.
Select in Kabat database the human antibody sequence Consensus KI the highest with RFB4 mouse source sequence framework region homology and Consensus subIII respectively as the framework region of humanized antibody light chain and heavy chain, after people source framework region is connected with Shu Yuan CDR district, as initial humanized antibody hRFB4 sequence, concrete comparison result as shown in Figure 1.
Use BLAST method, take PDB as searching database, the light chain to antibody after humanization and sequence of heavy chain carry out homology analysis respectively, according to result for retrieval, apply respectively 2UZI/L, H; 3EYP/C, D; 3KYM/A, B is stay in place form, and humanized hRFB4 antibody is built through the accurate three-dimensional model homology of row, builds result as shown in Figure 2.
The model having built is carried out to structural analysis, finally determine the aminoacid sequence of 5 humanized antibody hRFB4 albumen, as shown in YQ22-1 to YQ22-5.
The structure of embodiment 2, YQ22-1 weight chain expression vector
Entrust the variable region DNA sequence dna of the synthetic YQ22-1 light chain of Shanghai Jierui Biology Engineering Co., Ltd and heavy chain.Design PCR primer, increase respectively light chain of antibody and heavy chain gene, be then subcloned into expression vector pCDNA3.1 (Invitrogen company) with EcoRI, PmlI and SalI, PmlI double digestion respectively upper,
Large upgrading grain is prepared transfectional cell and is prepared humanized antibody.Its plasmid map as shown in Figure 3.
The expression and purification of embodiment 3, YQ22-1 antibody
1, YQ22-1 weight chain gene cotransfection
Transfection the day before yesterday, counting observation of cell activity, with 6～7 × 10
5the cell density of/ml is inoculated into 293E cell in 28ml FreeStyle 293 Expression Medium.Second day, cell mass concussion is broken up, and sample thief adds the blue counting of platform phenol observation of cell activity, viable count accounts for more than 90%, carries out transfection.30 μ g plasmids and 1ml Opti-MEMI are mixed, meanwhile, by 60 μ l Invitrogen 293 Fectin
tMreagent and 1ml Opti-MEMI mix.Room temperature leaves standstill 5min.Then the Opti-MEMI that is mixed with plasmid is joined in the Opti-MEMI that is mixed with transfection reagent, mix, obtain altogether 2ml mixed solution.Room temperature leaves standstill 20 minutes.Meanwhile, by 3 × 10
7individual cell is placed in 28ml FreeStyle 293 Expression Medium, adds above-mentioned 2ml mixed solution, after jog mixes, and in 37 ℃, 8%CO
2, 125rpm shaking table is cultivated 3-5 days.
2, the purifying of YQ22-1 antibody
The cell conditioned medium of collection is prepared to carry out protein purification, use the proteinA purifying YQ22 monoclonal antibody of GE company.Concrete steps are as follows: first, the Protein A being stored in 20% ethanol is shaken up, therefrom draw 2ml and add every pillar, make the glue of precipitation formation 1ml in every pillar.The reusable suitable number of times of pillar installing.Then, first, to the water that adds 5～10 times of column volumes in pillar, it is drained off under action of gravity, to wash residual ethanol off, then add the 1 × PB (0.02M, PH7.0) of 10 times of column volumes and make equally it drain off.The cell conditioned medium of collecting is first filtered with the filter of 0.45um, then adds the 10 × PB (0.2M, PH7.0) of 1/10th supernatant volumes, make the pH value of sample solution and ionic strength and binding buffer liquid phase ought, finally mix.The sample solution of handling well is added in post, make it under action of gravity, flow through pillar.Xiang Zhuzhong adds 1 × PB of 10～15 times of column volumes, and it is drained off, to wash foreign protein and unconjugated albumen off.After the PB in post drains off, carry out wash-out with the Glycine-Hcl damping fluid of the PH2.7 of 0.1M.First add 0.6ml, to wash PB residual in pillar off, do not collect and spill liquid.Add 3～4ml, gradation is collected and is spilt liquid again.Because protein is unstable under acidic condition, so will neutralize collected albumen.Solution used is the Tris-HCl damping fluid of the PH8.8 of 1.5M.
3, the structural identification of YQ22-1 antibody
Get 2 μ g hRFB4 antibody, add respectively reduction and non-reduced protein electrophoresis loading 5 × loading buffer, loading cumulative volume is 20 μ l.The SDS-PAGE electrophoresis result of humanized antibody as shown in Figure 4, can be found out from glue figure, an albumen master tape of irreducibility electrophoresis result demonstration, and antibody collection of illustrative plates is clear, and its molecular size range is consistent with expection, is positioned at 150KD left and right.And under reductibility protein electrophoresis condition, the disulfide linkage of antibody molecule weight interchain is reduced agent and opens, should see respectively the electrophoretic band of light chain and heavy chain.Two protein electrophoresis bands of reductibility electrophoresis result clear display, size lays respectively at 50KD and 25KD left and right, consistent with expected theoretical value.
4, the quantitative analysis of YQ22-1 antibody
Determination of protein concentration adopts lorry method.First the standard protein BSA of 1mg/ml is diluted to 100 μ g/ml, then configure respectively the each 1ml of protein standard substance of 0 μ g/ml, 20 μ g/ml, 40 μ g/ml, 60 μ g/ml, 80 μ g/ml and 100 μ g/ml, prepare the each 1ml of testing protein sample of different weaker concns simultaneously.Configuration alkaline copper solution, each detectable level need add 5ml alkaline copper solution, shakes up rear room temperature and leaves standstill 10 minutes.In each detection sample, add 500ul phenol reagent fast, after shaking up rapidly, room temperature leaves standstill 30 minutes, under 650nm wavelength, measures absorbance value.Antibody protein concentration is 1.07mg/ml, as shown in Figure 5.
The evaluation of embodiment 4, YQ22-1 antibody biological function
1, the mensuration of hRFB4 affinity of antibody
Affinity of antibody is the important parameter of reflection antibody and antigenic binding property, therefore, measures the avidity of humanization hRFB4 antibody, and compares with the avidity of its mouse source parental antibody RFB4, and be the important indicator that judges the success or not of humanization process.
Adopt the affinity of antibody detection method based on ELISA method, respectively hRFB4 and RFB4 antibody are carried out to avidity mensuration, CD22 antigen is respectively with 0.5 μ g/ml and 1 μ g/ml coated elisa plate, take the RFB4 antibody of different concns as primary antibodie, take goat anti-mouse IgG-HRP traget antibody as two anti-, detect OD450/630 value.The EC of two kinds of coated concentration
50value is respectively 1.17 × 10
-5, 7.72 × 10
-6; Take the hRFB4 antibody of different concns as primary antibodie, take the anti-human IgG-HRP traget antibody of goat as two anti-, detect OD450/630 value.The EC of two kinds of coated concentration
50value is respectively 1.28 × 10
-5, 8.22 × 10
-6.And be 2.08 × 10 according to the equilibrium dissociation constant KD:RFB4 antibody of two kinds of antibody of formula calculating
-10, hRFB4 is 2.33 × 10
-10.Detected result as shown in Figure 6.
Result shows, hRFB4 compares with its mouse source parental antibody RFB4, and KD value raises to some extent, but notable difference not between the two shows that hRFB4 antibody has the avidity close with mouse source parental antibody.
As a rule, adopt CDR implantation technique to carry out after humanization mouse source antibody, can significantly reduce affinity of antibody, cause KD value to raise, therefore, after antibody humanization, conventionally need to carry out external affinity maturation process.HRFB4 of the present invention is close with parental antibody avidity, thereby can remove the process of it being carried out to external affinity maturation from, has greatly saved experimental period and energy.
2, the combination of hRFB4 antibody and cell surface CD22 antigen test
CD22 antigen has expression on many lymphocytic surfaces, as Ramous, Raji and Daudi etc.Whether the hRFB4 antibody that therefore, we detect after humanization can combine from the CD22 antigen on different cell strains surface.Thereby whether the humanized antibody of verifying us has the biologic activity combining with CD22 antigen.
The Ramous of equal amts, Raji and Daudi cell are hatched altogether with hRFB4, RFB4, T1h and PBS respectively, and wherein the concentration of antibody is 10 μ g/ml.With the positive contrast of mouse source parental antibody RFB4, the homotype contrast that T1h is human antibody, the negative contrast of PBS.Then respectively with detecting through row with the fluorescently-labeled antibody anti-human or anti-mouse IgG of FITC, result as shown in Figure 7.Show that hRFB4 all can be combined with the target antigen CD22 of three kinds of cell surfaces with RFB4 antibody, and homotype contrast T1h and PBS all can not be in conjunction with.
3, the detection of hRFB4 antibody cell internalization effect
After RFB4 antibody is combined with the CD22 of cell surface antigen, can cause the internalization of immune complex, we claim that this phenomenon is the cell internalizing effect that antibody relies on.At present, many antitumor drugs take CD22 as target spot, in order further to strengthen the lethal effect of medicine to tumour cell, tend to select to utilize CD22 antibody to there is this feature of internalization effect, by itself and radioactive substance or the coupling of toxin phase, there is the more immunotoxin of powerful antitumor drug effect thereby be built into.Therefore the CD22 cell internalizing effect that, antibody causes is that the important biomolecule of the antibody take CD22 as target spot is learned feature.
By Daudi cell be placed in respectively 37 ℃ with 0 ℃ of two kinds of different condition under, then add hRFB4 antibody at 0 minute, 30 minutes and 1 hour three different time point respectively, antibody concentration is 10 μ g/ml.Then use the antibody amount of the anti-human IgG-FITC antibody test of goat cell surface combination.
Result as shown in Figure 8, under 37 ℃ of conditions, can obviously be observed the fluorescent signal causing due to antigen-antibody complex internalization and reduce.Show that RFB4 antibody can cause obvious internalization effect at 37 ℃.But under 0 ℃ of condition, cell internalizing activity inhibited.
HRFB4 antibody also shows the biological effect similar to mouse source parental antibody.In the time of 37 ℃, the fluorescent signal value detecting is low, shows to cause obvious internalization effect, thereby causes the hRFB4 antibody that is combined in cell surface to reduce.And the speed of this internalization effect is very high, the internalization effect of RFB4 and two kinds of antibody of hRFB4 all reached plateau in 30 minutes, with document (Du, X., Beers, R., et al, Cancer research 68,6300-6305 (2008) .) report unanimously.
Claims (10)
1. the monoclonal antibody of a specific binding people CD22, formed by variable region and constant region, the framework region in the CDR that variable region comprises mouse source and people source, it is characterized in that, the variable region of heavy chain of described antibody is as SEQ ID NO:1 composition, and the variable region of light chain of described antibody is respectively by SEQ ID NO:2,3,4,5,6 compositions.
2. monoclonal antibody according to claim 1, is characterized in that, the constant region of described antibody is any one of human IgG1, IgG2, IgG3 or IgG4.
3. monoclonal antibody according to claim 2, is characterized in that, the constant region of described antibody is human IgG1.
4. according to the monoclonal antibody described in claim 1-3 any one, it is characterized in that, described antibody is the humanized antibody that CDR transplants.
5. polynucleotide sequence, its monoclonal antibody as described in any one in claim 1-3 of encoding.
6. an expression vector, described carrier comprises polynucleotide sequence claimed in claim 5, and described carrier is pCDNA3.1.
7. a host cell, described host cell is via carrier transfection claimed in claim 6.
8. prepare the preparation method of monoclonal antibody described in claim 1-4 any one for one kind, described method comprises: the DNA expression vector transfection host cell that uses the described monoclonal antibody of coding, the cell culture supernatant that acquisition contains described antibody, and prepare target protein through Protien A column purification.
9. in claim 1-4, the monoclonal antibody of any one is for the preparation for the treatment of by the purposes in the medicine of the cell-mediated disease of expression CD22, and described is malignant lymphoma and autoimmune disorder by the cell-mediated disease of expressing CD22.
10. the purposes of monoclonal antibody according to claim 9, described malignant lymphoma refers to non-Hodgkin lymphoma, described autoimmune disorder is systemic lupus erythematous.
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