CN109651509A - The humanization monoclonal antibody and its preparation of anti-CD20 - Google Patents

Abstract

The present invention provides the humanization monoclonal antibodies and its preparation of anti-CD20.Specifically, the present invention provides a kind of new Humanized anti-CD 20 antibody.Antibody of the invention can combine CD20 antigen with high specificity, and affinity and bioactivity and low immunogenicity, stable structure, druggability with higher are good.And stability of the humanized antibody of the present invention in antibody preparation of the present invention is very good, is used to prepare prevention or treats the drug of the relevant disease of CD20.

Description

The humanization monoclonal antibody and its preparation of anti-CD20

Technical field

The present invention relates to field of medicaments, more particularly to the humanization monoclonal antibody and its preparation of anti-CD20.

Background technique

Humanized antibody refers to is further reduced source of mouse ingredient on the basis of chimeric antibody, and retains the anti-CDR region of mouse, The replacement of its part employment antibody moiety, in disease treatment, why humanized antibody is better than source of mouse antibody, is because in antibody The reduction of mouse ingredient can reduce the immunological rejection of body, another advantage of humanized antibody be it in vivo Long half time, the half-life period of source of mouse antibody less than one day, and humanized antibody up to a couple of days sometimes even longer time.

Reacted since mouse monoclonal antibody can cause human anti-mouse antibody in clinical treatment (human anti-mouse antibody, HAMA it), therefore on clinical treatment is restricted.

Therefore, there is still a need for the Humanized anti-CD 20 antibody that exploitation is suitable for treating patient for this field.

Summary of the invention

The purpose of the present invention is carry out humanization design and expression, acquisition and mouse to source of mouse CD20 antibody (Rituximab) In the humanized antibody of an order of magnitude, what utmostly reduction source of mouse antibody may cause in human body exempts from source affinity of antibody Epidemic focus.

There is provided a kind of CD20 humanized antibody of high-affinity high bioactivity and its applications for purpose again by the present invention.

The first aspect of the present invention, provides a kind of heavy chain variable region of antibody, the antibody heavy chain variable region include with Lower three complementary determining region CDR:

CDR1 shown in SEQ ID NO.:1,

CDR2 shown in SEQ ID NO.:2, and

CDR3 shown in SEQ ID NO.:3,

Also, heavy chain variable region shown in the affinity of the heavy chain variable region and the SEQ ID NO.:7 of source of mouse is affine Power is in an order of magnitude.

In another preferred example, the heavy chain variable region is corresponding to the selected from the group below of sequence shown in SEQ ID NO.:7 One or more amino acid mutate:

5th glutamine (Q), the 7th proline (P), the 11st leucine (L), the 12nd valine (V), the 20th methionine (M), the 38th lysine (K), the 43rd arginine (R), the 48th isoleucine (I), the 67th lysine (K), the 68th alanine (A), the 70th leucine (L), the 74th lysine (K), 76th serine (S), the 79th alanine (A), the 82nd glutamine (Q), the 87th threonine (T), 91 serines (S), the 113rd alanine (A).

In another preferred example, the heavy chain variable region of stating is selected from down corresponding to the mutation of sequence shown in SEQ ID NO.:7 Group:

Q5V、P7S、L11V、V12K、M20V、K38R、R43Q、I48M、K67R、A68V、L70M、L70I、K74T、S76T、 S76A, A79V, Q82E, T87R, S91T, A113Q, or combinations thereof.

In another preferred example, the heavy chain variable region is also corresponding to being selected from the group for sequence shown in SEQ ID NO.:7 Amino acid mutate:

44th glycine (G).

In another preferred example, the mutation that the heavy chain variable region corresponds to sequence shown in SEQ ID NO.:7 is further selected from down Group:

G44R。

In another preferred example, the heavy chain variable region is selected from the group:

(1) sequence heavy chain variable region as shown in SEQ ID NO.9 or 11；

(2) by amino acid sequence shown in SEQ ID NO.9 or 11 by least one (such as 1-20, preferably 1-15 A, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residue substitution, lack, repair It adorns and/or adds and formed, the sequence as shown in SEQ ID NO.9 or 11 with (1) described heavy chain variable region function is derivative Heavy chain variable region.

In another preferred example, the heavy chain variable region is in the framework region for corresponding to sequence shown in SEQ ID NO.:9 or 11 Can carrying out one or more, (1-20 is a, preferably 1-15 is a, more preferably 1-10 is a, more preferably 1-8 is a, more preferably 1-3 is a, most Good 1 or 2, ground) mutation of amino acid.

In another preferred example, the sequence of the heavy chain variable region is as shown in SEQ ID NO.:9 or 11.

Second aspect of the present invention provides a kind of heavy chain of antibody, and the heavy chain has as described in the first aspect of the invention Heavy chain variable region.

In another preferred example, the heavy chain of the antibody further includes heavy chain constant region.

In another preferred example, the heavy chain constant region is source of people, source of mouse or rabbit source, preferably source of people.

Third aspect present invention provides a kind of light chain variable region of antibody, and the antibody's light chain variable region includes following three A complementary determining region CDR:

CDR1 shown in SEQ ID NO.:4,

CDR2 shown in SEQ ID NO.:5, and

CDR3 shown in SEQ ID NO.:6,

Also, light chain variable region shown in the affinity of the light chain variable region and the SEQ ID NO.:8 of source of mouse is affine Power is in an order of magnitude.

In another preferred example, the light chain variable region is corresponding to the selected from the group below of sequence shown in SEQ ID NO.:8 One or more amino acid mutate:

1st glutamine (Q), the 3rd valine (V), the 5th serine (S), the 9th alanine (A), the 10th isoleucine (I), the 15th proline (P), the 17th glutamic acid (E), the 18th lysine (K), the 21st methionine (M), the 35th phenylalanine (F), the 41st serine (S), the 42nd serine (S), the 45th proline (P), the 46th tryptophan (W), the 59th valine (V), the 70th tyrosine (Y), 71st serine (S), the 76th arginine (R), the 77th valine (V), the 82nd alanine (A), the 103rd The leucine (L) of position.

In another preferred example, the mutation that the light chain variable region corresponds to sequence shown in SEQ ID NO.:8 is selected from down Group:

Q1D、V3Q、S5T、A9S、I10F、P15V、E17D、K18R、M21I、F35Y、S41K、S42A、P45L、W46L、 V59S, Y70F, S71T, R76S, V77L, A82F, L103V, or combinations thereof.

In another preferred example, the light chain variable region is selected from the group:

(1) sequence light chain variable region as shown in SEQ ID NO.10；

(2) by amino acid sequence shown in SEQ ID NO.10 by least one (such as 1-20, preferably 1-15, More preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residue substitution, missing, modification And/or addition and formed, with (1) described light chain variable region function the sequence as shown in SEQ ID NO.10 derived from light chain Variable region.

In another preferred example, the light chain variable region correspond to the framework region of sequence shown in SEQ ID NO.:10 can (1-20 is a, preferably 1-15 is a, more preferably 1-10 is a, more preferably 1-8 is a, more preferably 1-3 is a, most preferably for progress one or more Ground 1 or 2) amino acid mutation.

In another preferred example, the sequence of the light chain variable region is as shown in SEQ ID NO.:10.

Fourth aspect present invention provides a kind of light chain of antibody, and the light chain has as described in third aspect present invention Light chain variable region.

In another preferred example, the light chain of the antibody further includes constant region of light chain.

In another preferred example, the constant region of light chain is source of people, source of mouse or rabbit source, preferably source of people.

Fifth aspect present invention provides a kind of antibody, and the antibody includes

(1) heavy chain variable region as described in the first aspect of the invention；And/or

(2) light chain variable region as described in the third aspect of the present invention；

In another preferred example, the antibody includes heavy chain as described in respect of the second aspect of the invention；And/or such as the present invention Light chain described in fourth aspect.

In another preferred example, the antibody has the heavy chain variable region as shown in SEQ ID NO.9 or 11；And/or such as Light chain variable region shown in SEQ ID NO.10.

In another preferred example, the light-chain variable sequence of the antibody is as shown in SEQ ID NO.10；And/or it is described anti- The weight chain variabl area sequence of body is as shown in SEQ ID NO.9 or 11.

In another preferred example, the light-chain variable sequence of the antibody is as shown in SEQ ID NO.10；And it is described anti- The weight chain variabl area sequence of body is as shown in SEQ ID NO.9.

In another preferred example, the antibody has sequence light chain variable region as shown in SEQ ID NO.10；And institute Antibody is stated with sequence heavy chain variable region as shown in SEQ ID NO.11.

In another preferred example, the antibody is humanized antibody.

In another preferred example, the antibody is specific binding CD20.

In another preferred example, the antibody is double-chain antibody or single-chain antibody.

In another preferred example, the antibody is monoclonal antibody.

In another preferred example, the antibody is bispecific antibody.

In another preferred example, the antibody is drug conjugates form.

Sixth aspect present invention provides a kind of recombinant protein, and the recombinant protein includes

(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention Antibody；And

(ii) sequence label of optional assistance expression and/or purifying.

In another preferred example, the sequence label includes 6His label.

In another preferred example, the recombinant protein (or polypeptide) includes fusion protein.

In another preferred example, the recombinant protein is monomer, dimer or polymer.

Seventh aspect present invention provides a kind of antibody preparation, and the antibody preparation includes:

(a) antibody as described in fifth aspect present invention；And

(b) carrier, the carrier include: buffer, sterile water, optional surfactant.

Eighth aspect present invention provides a kind of kit, and the kit contains and resists described in seventh aspect present invention Body preparation, and contain the container of the antibody preparation.

Ninth aspect present invention provides a kind of CAR construction, the scFv of the antigen binding regions of the CAR construction Section is to specifically bind to the combined area of CD20, and the scFv has heavy chain variable region as described in the first aspect of the invention With light chain variable region as described in the third aspect of the present invention.

Tenth aspect present invention provides a kind of immunocyte of recombination, the immunocyte expression external source such as this hair CAR construction described in bright 9th aspect.

In another preferred example, the immunocyte is selected from the group: NK cell, T cell.

In another preferred example, the immunocyte comes from people or non-human mammal (such as mouse).

Tenth one side of the invention provides a kind of antibody drug conjugates, and the antibody drug conjugates contain:

(a) antibody moiety, the antibody moiety are selected from the group: heavy chain variable region as described in the first aspect of the invention, such as Heavy chain described in second aspect of the present invention, light chain variable region as described in the third aspect of the present invention, such as fourth aspect present invention institute Antibody described in the light chain or fifth aspect present invention stated, or combinations thereof；With

(b) coupling moiety being coupled with the antibody moiety, the coupling moiety are selected from the group: detectable marker, medicine Object, toxin, cell factor, radionuclide, enzyme, or combinations thereof.

In another preferred example, the antibody moiety and the coupling moiety are carried out even by chemical bond or connector Connection.

The twelfth aspect of the present invention provides a kind of purposes of active constituent, and the active constituent is selected from the group: such as this hair It is heavy chain variable region described in bright first aspect, heavy chain as described in respect of the second aspect of the invention, as described in the third aspect of the present invention Antibody described in light chain variable region, light chain as described in the fourth aspect of the present invention or fifth aspect present invention, such as present invention the 6th Recombinant protein described in aspect, the immunocyte as described in tenth aspect present invention, resisting as described in the tenth one side of the invention Body drug conjugates, or combinations thereof, the active constituent is used for

(a) detection reagent or kit are prepared；

(b) drug or preparation of preparation prevention and/or treatment CD20 related disease；And/or

(c) drug or preparation of preparation prevention and/or treating cancer or tumour.

In another preferred example, the tumour is selected from the group: neoplastic hematologic disorder, solid tumor, or combinations thereof.

In another preferred example, the neoplastic hematologic disorder is selected from the group: acute myelocytic leukemia (AML), multiple marrow Tumor (MM), chronic lymphocytic leukemia (CLL), acute lymphatic leukaemia (ALL), diffusivity large B cell lymphoid tumor (DLBCL), Hodgkin lymphoma, or combinations thereof.In another preferred example, the solid tumor is selected from the group: gastric cancer, gastric cancer peritoneum Transfer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, palace Neck cancer, oophoroma, lymph cancer, nasopharyngeal carcinoma, adrenal tumor, tumor of bladder, non-small cell lung cancer (NSCLC), glioma, son Endometrial carcinoma, or combinations thereof.

In another preferred example, the tumour is the tumour of high expression CD20.

In another preferred example, the drug or preparation are used to prepare prevention and/or treatment and CD20 (the expression positive) The drug or preparation of relevant disease.

In another preferred example, the antibody is drug conjugates (ADC) form.

In another preferred example, the detection reagent or kit are for diagnosing CD20 related disease.

In another preferred example, the detection reagent or kit are for CD20 albumen in test sample.

In another preferred example, the detection reagent is detection lug.

The 13rd aspect of the present invention provides a kind of pharmaceutical composition, and the pharmaceutical composition contains:

(i) active constituent, the active constituent are selected from the group: heavy chain variable region as described in the first aspect of the invention, such as Heavy chain described in second aspect of the present invention, light chain variable region as described in the third aspect of the present invention, such as fourth aspect present invention institute Antibody described in the light chain or fifth aspect present invention stated, the recombinant protein as described in sixth aspect present invention, such as present invention the Immunocyte described in ten aspects, the antibody drug conjugates as described in tenth one side of the invention, or combinations thereof；And

(ii) pharmaceutically acceptable carrier, diluent or excipient.

In another preferred example, the pharmaceutical composition is liquid formulation.

In another preferred example, the pharmaceutical composition is injection.

In another preferred example, in described pharmaceutical composition, the concentration of the cell is 1 × 10³-1×10⁹A cell/ Ml, preferably 1 × 10⁵-1×10⁸A cell/ml.

In another preferred example, described pharmaceutical composition also other drugs (such as core containing selective killing tumour cell Sour drug, antibody drug, target medicine, other immunocyte drugs, other CAR-T drugs, chemotherapeutics, or combinations thereof).

In another preferred example, the pharmaceutical composition is for treating tumour.

In another preferred example, the tumour is the tumour of high expression CD20.

Fourteenth aspect of the present invention provides a kind of polynucleotides, polynucleotide encoding polypeptide selected from the group below:

(1) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Described in light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or fifth aspect present invention Antibody；Or

(2) recombinant protein as described in sixth aspect present invention；

(3) the CAR construction as described in ninth aspect present invention.

The fifteenth aspect of the present invention provides a kind of carrier, and the carrier contains as described in fourteenth aspect of the present invention Polynucleotides.

In another preferred example, the carrier include: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.

The 16th aspect of the present invention provides a kind of genetically engineered host cell, and the host cell contains such as this It invents and integrates in carrier or genome described in the 15th aspect just like polynucleotides described in fourteenth aspect of the present invention.

The 17th aspect of the present invention provides a kind of method of preparation engineering immunocyte, comprising the following steps:

(A) immunocyte to be rebuilt is provided；With

(B) the first expression cassette is imported into the immunocyte to be rebuilt, wherein first expression cassette expression is as originally CAR construction described in the 9th aspect is invented, to obtain the immunocyte of engineering.

In another preferred example, first expression cassette contains CAR construction described in coding ninth aspect present invention Nucleic acid sequence.

In another preferred example, first expression cassette is located on carrier or is incorporated into the immunocyte of the engineering Chromosome in.

In another preferred example, the carrier is selected from the group: DNA, RNA, plasmid, slow virus carrier, adenovirus vector, Retroviral vector, transposons, oncolytic virus carrier, other gene transfer systems, or combinations thereof.

In another preferred example, the carrier is viral vectors (such as slow virus carrier).

In another preferred example, the carrier is transposon vector.

In another preferred example, the immunocyte is T cell or NK cell.

In another preferred example, the method further includes carrying out function and validity to the engineering immunocyte of acquisition The step of detection.

The 18th aspect of the present invention provides a kind of method of CD20 albumen in vitro detection sample, and the method includes steps It is rapid:

(1) in vitro, the sample is contacted with the antibody as described in fifth aspect present invention；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are CD20 eggs in sample It is white.

In another preferred example, the method is non-diagnostic and non-treatment.

The 19th aspect of the present invention provides a kind of detection plate, and the detection plate includes: substrate (support plate) and test Item, the test-strips contain the antibody as described in fifth aspect present invention or the antibody medicine as described in the tenth one side of the invention Object conjugate.

The 20th aspect of the present invention provides a kind of kit, and the kit includes:

The first container, and the first nucleic acid sequence in the first container, first nucleic acid sequence contain expression originally Invent the first expression cassette of CAR construction described in the 9th aspect.

20th one side of the invention provides a kind of diagnostic kit, comprising:

(1) the first container contains antibody described in fifth aspect present invention in the first container；And/or

(2) second container, the secondary antibody containing antibody described in anti-fifth aspect present invention in the second container.

In another preferred example, the kit contains detection plate described in the 19th aspect of the present invention.

The 22nd aspect of the present invention provides a kind of preparation method of recombinant polypeptide, which comprises

(a) under conditions suitable for the expression, the host cell as described in terms of cultivating such as the present invention the 16th；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is anti-as described in fifth aspect present invention Body or the recombinant protein as described in sixth aspect present invention.

The 23rd aspect of the present invention provides a kind of method for treating disease relevant to CD20, which comprises The antibody as described in fifth aspect present invention, the recombinant protein as described in sixth aspect present invention, this hair are applied to the object of needs CAR construction described in bright 9th aspect, immunocyte described in tenth aspect present invention, described in the tenth one side of the invention Pharmaceutical composition described in antibody drug conjugates, the 13rd aspect of the present invention.

In another preferred example, the disease relevant to CD20 includes the cancer or tumour of the CD20 positive.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows different humanized whole antibodies' flow cytometer detection figures.

Fig. 2 shows the electrophoretogram of entirely anti-expression and purification.

Fig. 3 shows the alignment of humanized antibody NH2-1-NL2-1 Yu source of mouse antibody heavy chain variable region.

Fig. 4 shows the alignment of humanized antibody NH2-1-NL2-1 Yu source of mouse antibody's light chain variable region.

Fig. 5 shows the alignment of humanized antibody NH1-1-NL2-1 Yu source of mouse antibody heavy chain variable region.

Fig. 6 shows the alignment of humanized antibody NH1-1-NL2-1 Yu source of mouse antibody's light chain variable region.

Fig. 7 shows the source of mouse antibody of various concentration and the flow analysis chart of Raji cell combination.

Fig. 8 shows the humanization NH1-1-NL2-1 antibody of various concentration and the flow analysis chart of Raji cell combination.

Fig. 9 shows the humanization NH2-1-NL2-1 antibody of various concentration and the flow analysis chart of Raji cell combination.

Figure 10 shows 3 kinds of antibody of various concentration and the average fluorescent strength statistical chart of Raji cell combination.

Specific embodiment

The present inventor is by extensive and in-depth research, by largely screening, unexpectedly obtains a kind of excellent with affinity The Humanized anti-CD 20 antibody of different and good structural stability.Specifically, skeleton area of the present invention to human antibody template Carry out humanization.Antibody after humanization reach with affinity similar in source of mouse antibody, be an order of magnitude.

Specifically, the present invention has carried out humanization design and transformation to source of mouse CD20 antibody (Rituximab), and expresses Corresponding humanized antibody is verified through overflow-type verifying and affinity, obtains 2 and the consistent source of people of source of mouse affinity of antibody Change antibody sequence, and further screen, be finally sieved to one plant it is consistent with source of mouse affinity of antibody and reach an order of magnitude Humanized antibody sequence (Ruhab-NL2-1-NH1-1).The present invention is completed on this basis.

Term

In order to be easier to understand the present invention, certain technical and scientific terms are defined in detail below.Unless another herein It explicitly defines, all other technical and scientific term used herein all has those skilled in the art of the art Normally understood meaning.

Amino acid three-letter codes used in the present invention and single letter code such as J.biol.chem, in 243, p3558 (1968) It is described.

As used herein, term " giving " and " processing " refer to exogenous drugs, therapeutic agent, diagnosticum or composition application In animal, people, subject, cell, tissue, organ or biofluid." giving " and " processing " can refer to that treatment, drug metabolism are dynamic Mechanics, diagnosis, research and experimental method.The contact of cell handled including contact and reagent of the reagent with cell with fluid, Contact of the fluid with cell." giving " and " processing " still means that through reagent, diagnosis, combining compositions or passes through another cell External and ex vivo treatment." processing " refers to treatment processing, prevention or preventative when being applied to people, animal or study subject Measure, research and diagnosis；Including anti-CD 20 antibodies and human or animal, subject, cell, tissue, physiological compartment or physiological fluid Contact.

As used herein, term " treatment ", which is showed, gives the interior or topical therapeutic agent of patient, includes any one of the invention Anti-CD 20 antibodies and combinations thereof, the patient has one or more disease symptoms, and the known therapeutic agent is to these diseases Shape has therapeutic effect.In general, being given so that the amount of the therapeutic agent of one or more disease symptoms (therapeutically effective amount) is effectively relieved Patient.

As used herein, term " optional " or " optionally " mean event or situation described later can occur but It is not required to occur.For example, " optionally including 1-3 antibody heavy chain variable region " refers to that the antibody heavy chain variable region of particular sequence can To have but be not required, 1,2 or 3 can be.

" sequence identity " of the present invention indicates in the case where having the mutation such as replacement, insertion or missing appropriate Optimal comparison and when comparing, the identity degree between two nucleic acid or two amino acid sequences.Heretofore described sequence Sequence identity between its sequence with identity can be at least 85%, 90% or 95%, more preferably at least 95%. Non-limiting embodiment includes 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

CD20

CD20 is expressed in the surface of the B cell in each stage of the Development And Differentiation in addition to thick liquid cell, overregulates cross-film calcium ionic current It is dynamic directly to work to B cell, important adjustment effect is played in B cell proliferation and differentiation.CD20 antigen is a kind of B cell point To change antigen, is only located at pre B cell and mature B cell, it is expressed in 95% or more B cell lymthoma, and in Hematopoietic Stem It is not expressed in cell, plasma cell and other normal tissues.Therefore CD20 is the reason of targeted therapy B cell lymphoma and leukaemia Think target spot.

Source of mouse Rituximab variable region sequences information

The light chain variable region (SEQ ID NO.8) of source of mouse Rituximab monoclonal antibody

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTS YSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTV

The heavy chain variable region (SEQ ID NO.7) of source of mouse Rituximab monoclonal antibody

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKAT LTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA

Humanization NL2-1-NH1-1 variable region sequences information

Humanization monoclonal antibody NL2-1-NH1-1 light chain variable region (SEQ ID NO.10)

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTS FTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV

Humanization monoclonal antibody NL2-1-NH1-1 heavy chain variable region (SEQ ID NO.9)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRVT ITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS

Humanization Ruhab-NL2-1-NH2-1 variable region sequences information

Humanization monoclonal antibody Ruhab-NL2-1-NH2-1 light chain variable region (SEQ ID NO.10)

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTS FTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV

Humanization monoclonal antibody Ruhab-NL2-1-NH2-1 heavy chain variable region (SEQ ID NO.11)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQGLEWMGAIYPGNGDTSYNQKFKGRVT MTADTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS

Antibody

As used herein, term " antibody " refers to immunoglobulin, is by two identical heavy chains and two identical light chains Four peptide chain structures being formed by connecting by interchain disulfide bond.The amino acid of immunoglobulin heavy chain constant region forms and puts in order Difference, therefore its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or be the isotype of immunoglobulin, i.e., IgM, IgD, IgG, IgA and IgE, corresponding heavy chain are respectively μ chain, δ chain, γ chain, α chain and ε chain.Same class Ig is according to it Compared with the difference of the number and location of sequence amino acid composition and heavy chain disulfide bond, and different subclass can be divided into, as IgG can be divided into IgG1, IgG2, IgG3, IgG4.Light chain is divided into κ chain or λ chain according to the difference of constant region.Every class Ig can have κ in five class Ig Chain or λ chain.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art.

Antibody light chain of the present invention can further include constant region of light chain, the constant region of light chain include source of people or κ, λ chain or its variant of source of mouse.

In the present invention, heavy chain of antibody of the present invention can further include heavy chain constant region, the light chain constant Area includes IgG1, IgG2, IgG3, IgG4 or its variant of source of people or source of mouse.About 110 close to N-terminal of heavy chain of antibody and light chain The sequence variation of amino acid is very big, is variable region (area Fv)；Remaining amino acid sequence close to C-terminal is relatively stable, is constant region. The variable region skeleton area (FR) relatively conservative including 3 hypervariable regions (HVR) and 4 sequences.3 hypervariable regions determine the special of antibody Property, also known as complementarity-determining region (CDR).Every light chain variable region (LCVR) and heavy chain variable region (HCVR) by 3 CDR regions and 4 FR district's groups are at from aminoterminal to the sequence sequence completing cardinal extremity and being arranged successively are as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.3 CDR regions of light chain refer to LCDR1, LCDR2 and LCDR3；3 CDR regions of heavy chain refer to HCDR1, HCDR2 and HCDR3.

Term " source of mouse antibody " is anti-for the monoclonal of the anti-CD20 prepared according to this field knowledge and skills in the present invention Body.With CD20 antigen injection subjects when preparation, then separation expression has the miscellaneous of the antibody of required sequence or functional characteristic Hand over tumor.In a preferred embodiment of the present invention, the source of mouse CD20 antibody or its antigen-binding fragment, can be further Constant region of light chain comprising source of mouse κ, λ chain or its variant, or further include the weight of mouse IgG 1, IgG2, IgG3 or its variant Chain constant region.

Term " chimeric antibody (chimeric antibody) " is by the constant of the variable region of murine antibody and human antibody Antibody made of area's fusion, can mitigate the immune response of murine antibody induction.

Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted Antibody), refer to the antibody variable region frame that the CDR sequence of mouse is transplanted to people, i.e., different types of human germline antibody's structure The antibody generated in frame sequence.Humanized antibody can overcome chimeric antibody due to carrying a large amount of mouse protein ingredients, to induce Heterologous reaction.Such frame sequence can be from public DNA database or disclosed ginseng including germline antibody gene sequences Examine document acquisition.While to avoid immunogenicity from declining, caused activity decline can be to the human antibody variable framework Sequence carries out minimum inverse transition or back mutation, to keep activity.

Term " antigen-binding fragment of antibody " (or referred to as " antibody fragment ") refer to that the holding specific binding of antibody is anti- One or more segments of the ability of former (for example, CD20).Oneself display can carry out the anti-of antibody using the segment of full length antibody Former binding function.The example for the binding fragment for including in term " antigen-binding fragment of antibody " includes

(i) Fab segment, the monovalent fragment being made of VL, VH, CL and CH1 structural domain；

(ii)F(ab’)₂Segment includes the bivalent fragment by two Fab segments compared with the disulphide bridges connection on sequence；

(iii) the Fd segment being made of VH and CH1 structural domain；

(iv) the Fv segment being made of VH the and VL structural domain of the single armed of antibody.

Fv antibody contains antibody heavy chain variable region, light chain variable region, but does not have constant region, and has whole antigen bindings position The minimum antibody fragment of point.In general, Fv antibody also includes the peptide linker between VH and VL structural domain, and it is capable of forming antigen In conjunction with required structure.

Term " CDR " refers to one of 6 hypervariable regions for mainly facilitating antigen binding in the variable domains of antibody.Described 6 One of most common definition of a CDR is by Kabat E.A et al., (1991) Sequences of proteins of Immunological interest.NIH Publication91-3242) it provides.

Term " epitope " or " antigenic determinant " refer to the position (example that immunoglobulin or antibody specificity combine on antigen Such as, the privileged site on CD20 molecule).Epitope includes usually at least 3,4,5,6,7,8,9,10,11 with unique space conformation, 12,13,14 or 15 amino acid continuously or discontinuously.

Term " specific binding ", " selective binding ", " selectively combining " and " specifically combining " refers to antibody Combination to the epitope on predetermined antigen.In general, antibody is approximately to be less than 10^-7M, such as approximately less than 1O^-8M、1O^-9M Or lO^-10M or smaller affinity (KD) combine.

Term " competitive binding " refer to on the extracellular region of monoclonal antibody identification CD20 of the invention same epitope ( Referred to as antigenic determinant) or same epitope a part and antibody with the antigen binding.With monoclonal antibody of the invention Antibody in conjunction with same epitope refers to identification and is incorporated into the amino acid sequence for the CD20 that monoclonal antibody of the invention is identified Antibody.

Term " KD " or " Kd " refer to specific antibodies-antigen interactions Dissociation equilibrium constant.In general, of the invention is anti- Body is to be less than about 10^-7M is, for example, less than about 10^-8M、10^-9M or l0^-10M or smaller Dissociation equilibrium constant (KD) combine CD20。

As used herein, term " antigenic determinant " refers to discontinuous on antigen, by antibody of the present invention or antigen binding fragment The three-dimensional space site of section identification.

The present invention not only includes complete antibody, further includes the segment or antibody and other sequences with immunocompetent antibody Arrange the fusion protein formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.

In the present invention, antibody includes mouse prepared by the technology known to those skilled in the art, chimeric, humanization Or full people antibody.Recombinant antibodies, such as chimeric and humanization monoclonal antibody, including people's and inhuman portion Point, DNA recombinant technique well known in the art can be used.

As used herein, term " monoclonal antibody " refers to the antibody of the clones secrete derived from individual cells source.Monoclonal Antibody is high degree of specificity, for single epitope.The cell may be eukaryon, protokaryon or bacteriophage gram Grand cell strain.

In the present invention, antibody can be monospecific, bispecific, tri-specific or more multiple specifics.

In the present invention, antibody of the invention further includes its conservative variant, is referred to and the amino acid sequence of antibody of the present invention Column are compared, and have at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid by property it is similar or Similar amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and produce It is raw.

Table 1

Initial residue	Representative substitution	It is preferred to replace
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Humanized anti-CD 20 antibody

The present invention provides Humanized anti-CD 20 antibody (hereinafter referred to as CD20 antibody).Specifically, the present invention provides a kind of needle The humanized antibody of high specific and high-affinity to CD20 comprising heavy chain and light chain, the heavy chain contain weight chain variable Area (VH) amino acid sequence, the light chain contain light chain variable region (VL) amino acid sequence.

Mouse monoclonal antibody heavy chain CDR is transplanted to human antibody heavy chain's skeleton area for the first time by Jones in 1986 et al., then with mouse monoclonal antibody Light chain is assembled into complete antibody and maintains affinity similar with former mouse monoclonal antibody, and the development for antibody humanization's technology provides Thinking.The method that Queen in 1989 et al. is transplanted by CDR, successfully constructs anti CD 25 humanized antibody, and this method uses Human antibody Eu skeleton area carries out humanization, and in skeleton area, moiety site remains source of mouse antibody amino acid to keep affinity. It is that template carries out CDR shifting that Presta in 1992 et al., which is reported with human antibody subgroup consensus sequence (consensus sequence), Plant the method for successfully constructing humanization.Pedersen in 1994 et al. reports the method with surface remodeling (resurfacing) To antibody humanization.Hsiao in 1994 et al. reports the source of people that CDR transplanting is carried out with the sequence skeleton area human antibody Germline Change method.Jespers in 1994 et al. successfully constructs humanization side with the method for phage library (shuffling library) Method.

In antibody humanization the selection in human skeleton area it is usual there are two types of, one is known mature antibody, and one is people Germline sequence.Known maturation antibody backbone area usually contains somatic mutation site, may bring potential immunogene Property.Compared to mature antibody, immunogenicity is lower on people's Germline sequence skeleton domain tyeory, and structure is more flexible, plastic Strong, the acceptant different CDR region of property.The frequency of use of human antibody Germline gene in human body has certain deviation Property, the antibody after selecting the high Germline skeleton area humanization of frequency of use has that immunogenicity is low, expression quantity is high, structure is steady The advantages that determining, therefore the present invention is in humanization and non-selected with the highest Germline sequence of source of mouse antibody similarity, but Similitude and human body frequency of use are taken into account, is screened by many experiments, the skeleton area of Rituximab has been selected to carry out humanization. The present invention selects human antibody Germline skeleton area to carry out CDR transplanting, and the humanized antibody structure constructed in this way is more stable, table Up to amount is high, immunogenicity is low, druggability is higher.

Specifically, as described in terms of first aspect present invention to the 5th:

Humanization monoclonal antibody NL2-1-NH1-1 light chain variable region (SEQ ID NO.10)

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTS FTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV

Humanization monoclonal antibody NL2-1-NH1-1 heavy chain variable region (SEQ ID NO.9)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRVT ITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS

Humanization Ruhab-NL2-1-NH2-1 variable region sequences information

Humanization monoclonal antibody Ruhab-NL2-1-NH2-1 light chain variable region (SEQ ID NO.10)

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTS FTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV

Humanization monoclonal antibody Ruhab-NL2-1-NH2-1 heavy chain variable region (SEQ ID NO.11)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQGLEWMGAIYPGNGDTSYNQKFKGRVT MTADTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS

In another preferred example, described by adding, lacking, modifying and/or replacing at least one amino acid sequence institute shape At sequence be preferably homology be at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% Amino acid sequence.

Antibody of the invention can be double-strand or single-chain antibody, and can be preferably full humanized antibody.

Antibody derivatives of the present invention can be single-chain antibody, and/or antibody fragment, such as: Fab, Fab ', (Fab ')₂、 Or in the field other known antibody derivatives etc. and IgA, IgD, IgE, IgG and IgM antibody or other hypotypes it is anti- Any one or a few in body.

Antibody of the present invention can be the antibody of the humanized antibody of targeting CD20, CDR grafting and/or modification.

In above content of the present invention, the addition, missing, modification and/or the amino acid quantity replaced, preferably no more than The 40% of initial amino acid sequence total amino acid quantity, more preferably less than 35%, more preferably 1-33%, more preferably 5- 30%, more preferably 10-25%, more preferably 15-20%.

The present invention is successfully made CD20 mouse monoclonal antibody humanization modified, and the antibody after humanization reaches and chimeric antibody phase Close affinity (up to an order of magnitude), passes through the Primary Study to the humanized antibody solubility and endogenous fluorescence, it was demonstrated that The humanization has had preliminary druggability, and there is the humanization monoclonal antibody medicine for being developed further into targeted therapy in future.

In the present invention, CDR1, CDR2, CDR3 of the heavy chain variable region or light chain variable region of humanized antibody of the invention Respectively as shown in horizontal line in Fig. 3-6.

The preparation of antibody

Any method for being suitable for generating monoclonal antibody can be used in generating CD20 antibody of the invention.For example, can use Animal is immunized in connection or naturally occurring CD20 albumen or its segment.It can be used suitable methods of vaccination, including adjuvant, Immunostimulant repeats booster immunization inoculation, and one or more approach can be used.

The CD20 of any suitable form all can serve as immunogene (antigen), special to CD20 inhuman anti-for generating Body screens the biological activity of the antibody.Immunogene can be used alone, or with it is known in the art one or more immune Originality reagents recombination uses.Immunogene can be purified by natural origin, or be generated in the cell of genetic modification.Coding is exempted from The DNA of epidemic focus can be (such as cDNA) of genome or non genome on source.Suitable genetic carrier table can be used Up to the DNA of encoding immunogens, the carrier includes but is not limited to adenovirus vector, baculovirus vector, plasmid and non-viral load Body.

Humanized antibody can be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA and IgE.Equally, Any sort light chain can use in the Compounds and methods for of this paper.Specifically, κ, λ chain or its variant are in change of the invention Closing can use in object and method.

The illustrative methods of humanization CD20 antibody of the present invention are described in embodiment 1.

The sequence of antibody of the present invention or the DNA molecular of its segment can use routine techniques, for example utilize PCR amplification or gene The methods of group library screening obtains.In addition, can also be fused together the coded sequence of light chain and heavy chain, single-chain antibody is formed.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.Then the DNA sequence dna can be introduced this In field in known various existing DNA moleculars (or such as carrier) and cell.

Term " nucleic acid molecules " refers to DNA molecular and RNA molecule.Nucleic acid molecules can be single-stranded or double-stranded but preferred It is double-stranded DNA.When nucleic acid and another nucleic acid sequence to be placed in functional relationship, nucleic acid is " effectively connecting ".For example, such as Fruit promoter or enhancer influence the transcription of coded sequence, then promoter or enhancer are operatively connected to the code sequence Column.

Term " carrier " is the nucleic acid molecules for referring to transport oneself another nucleic acid connected to it.In an embodiment In, carrier is " plasmid ", and other DNA section can be connected on circular double stranded DNA ring therein by referring to.

The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.

Term " host cell " refers to the cell for introducing expression vector thereto.It is thin that host cell can be protokaryon Born of the same parents, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as plant or zooblast (such as mammalian cell).

Heretofore described can be carried out the step of converting host cell with recombinant DNA with technology well known in the art.It obtains The transformant obtained can express the polypeptide of coded by said gene of the invention with conventional method culture, transformant.According to host used Cell is cultivated under suitable conditions with conventional medium.

In general, culture converts resulting host cell under conditions of being suitble to antibody expression of the present invention.Then with routine Immunoglobulin purification step, such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange layer The routine well known to those skilled in the art such as analysis, hydrophobic chromatography, sieve chromatography or affinity chromatography isolates and purifies means and purifies To antibody of the invention.

Gained monoclonal antibody can be identified with conventional means.For example, the binding specificity of monoclonal antibody is available immune Precipitating or external combine test (such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA)) to measure.

Antibody preparation

Antibody has different stability in different Formulation Buffers, shows as variation, the antibody of charge heterogeneity Degradation, polymerization of molecule etc., the variation of these quality properties and the physicochemical property of antibody itself are related, therefore, in antibody drug Development process need to screen the Formulation Buffer for being suitble to its own according to the physicochemical property of different antibodies.Currently used antibody system Agent buffer system has phosphate buffer, citrate buffer solution, histidine buffering liquid etc., while can be added not according to antibody characteristic The excipient such as salt ion or sorbierite, trehalose, sucrose with concentration and the surface-actives such as suitable polysorbas20 or Tween 80 Agent, to maintain the stability of antibody.

Antibody preparation of the present invention is as described in seventh aspect present invention.

The aggregate and precipitate of humanized antibody of the present invention, hydrolysis, oxygen can be effectively suppressed in antibody drug combination preparation of the invention Change and the side reactions such as deamidation, at the same can effectively improve product in pressurization (high temperature, strong illumination and freeze thawing etc.), accelerate and long-term Stability under refrigerated condition.

Pharmaceutical composition

The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains The antibody stated or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell and pharmaceutically acceptable load Body.In general, these substances can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH Usually about 5-8, preferably pH is about 6-8, although pH value can have with the property and illness to be treated for being formulated substance Changed.Prepared pharmaceutical composition can be administered by conventional route, including (but being not limited to): in tumor, In peritonaeum, intravenous or local administration.

Antibody of the present invention is also possible to express the cell therapy being used in the cell by nucleotide sequence, for example, institute Antibody is stated for Chimeric antigen receptor T cell immunotherapy (CAR-T) etc..

Pharmaceutical composition of the invention can be directly used for combining CD20 protein molecular, thus can be used for preventing and treating CD20 Relevant disease.In addition, also can be used simultaneously other therapeutic agents.

Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutically acceptable carrier or tax of the present invention Shape agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Drug system Agent should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile Under the conditions of manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg of about 1 microgram/kg body weight-daily Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.

It is that the pharmaceutical composition of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg weight, compared with Good ground dosage is about 20 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration way The factors such as diameter, patient health situation, within the scope of these are all skilled practitioners technical ability.

Detection applications and kit

Antibody of the invention can be used for detecting application, such as detecting sample, to provide diagnostic message.

In the present invention, used sample (sample) includes cell, tissue samples and biopsy specimen.The art that the present invention uses Language " biopsy " should include the biopsy of all kinds well known by persons skilled in the art.Therefore biopsy used in the present invention can wrap Include the tissue samples for example prepared by the puncture of endoscopic procedures or organ or needle puncture biopsy.

Sample used in the present invention includes fixed or preservation cell or tissue sample.

The present invention also provides a kind of kits for referring to and containing antibody (or its segment) of the invention, at of the invention one In preference, the kit further includes container, operation instructions, buffer etc..In preference, antibody of the invention can To be fixed on detection plate.

Main advantages of the present invention

(1) it is anti-up to the CD20 of the humanization of source of mouse antibody an order of magnitude to develop a kind of affinity for the first time by the present invention Body.

(2) present invention obtains a kind of CD20 antibody sequence of humanization for the first time.

(3) humanized antibody of the invention can reduce the immunological rejection that source of mouse CD20 antibody is generated in human body.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

The experiment of actual conditions is not specified in the embodiment of the present invention or test case, usually routinely condition carries out, or according to Raw material/goods manufacturer suggestion condition；The reagent in specific source is not specified, for the conventional reagent of market purchase.

Embodiment 1

One, experimental procedure 1, humanization design

Humanization design is carried out according to the amino acid sequence information (Rituximab) of CD20 heavy chain of antibody and light chain, is retained The CDR region sequence of original antibodies is constant, according to the result of germline alignment and antibody structure simulate as a result, weight Chain and light chain select different human antibody templates respectively, and the framework region after humanization carries out back mutation, if The following candidate humanized antibody sequence of meter.

Humanization candidate antibodies amino acid sequence information:

>NH1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRV TITRDTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:12)

>NH1-1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRV TITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:9)

>NH2

QVQLVQSGAEVKKPGASVKVSCKASGYTFTYNMHWVRQTPGQGLEWMGAIYPGNGDTSYNQKFKGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:13)

>NH2-1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQGLEWMGAIYPGNGDTSYNQKFKGRV TMTADTSTSTVYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:11)

>NH3

QVQLVQSGAEVKKPGASVKVSCKVSGYTFTSYNMHWVRQAPGKGLEWMGAIYPGNGDTSYNQKFKGRV TMTEDTSTDTAYMELSSLRSEDTAVYYCATSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:14)

>NH3-1

QVQLVQSGAEVKKPGASVKVSCKVSGYTFTSYNMHWVRQTPGKGLEWMGAIYPGNGDTSYNQKFKGRV TMTEDTSTDTAYMELSSLRSEDTAVYYCATSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:15)

>NL2-1

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGT SFTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV(SEQ ID NO.:10)

>NL3

EIVLTQSPATLSLSPGERATLSCRASSSVSYIHWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQWTSNPPTFGGGTKVEIKRTV(SEQ ID NO.:16)

Corresponding nucleic acid coding sequence information is as follows:

>NH1

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTCCGACAGACCCCTGGCCAGAGACTTGAATGGATG GGCGCCATCTATCCCGGCAACGGCGACACCTCCTACAACCAGAAATTCAAGGGCCGCGTGACCATCACCAGAGACA CATCTGCCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAG CACCTACTACGGCGGCGACTGGTACTTCAATGTGTGGGGCCAGGGCACCACCGTGACAGTTTCT(SEQ ID NO.: 17)

>NH1-1

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTCCGACAGACCCCTGGCCAGAGACTTGAATGGATG GGCGCCATCTATCCCGGCAACGGCGACACCTCCTACAACCAGAAATTCAAGGGCAGAGTGACCATCACCGCCGACA CATCTGCCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAG CACCTACTACGGCGGCGACTGGTACTTCAATGTGTGGGGCCAGGGCACCACCGTGACAGTTTCT(SEQ ID NO.: 18)

>NH2

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGCCAGCGGCTACACCTTTACCTACAACATGCACTGGGTCCGACAGACCCCTGGACAGGGACTTGAATGGATGGGC GCCATCTATCCCGGCAACGGCGACACCAGCTACAACCAGAAATTCAAGGGCCGCGTGACCATGACCAGAGACACCA GCACCTCCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAGCAC CTACTACGGCGGCGACTGGTACTTCAATGTGTGGGGCCAGGGCACCACCGTGACAGTTTCT(SEQ ID NO.:19)

>NH2-1

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTCCGACAGACACCTGGACAGGGACTCGAATGGATG GGCGCCATCTATCCCGGCAATGGCGACACCTCCTACAACCAGAAATTCAAGGGCAGAGTGACCATGACCGCCGACA CCAGCACAAGCACCGTGTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAG CACCTACTACGGCGGCGACTGGTACTTCAATGTGTGGGGCCAGGGCACCACCGTGACAGTTTCT(SEQ ID NO.: 20)

>NH3

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGTGTCCGGCTACACCTTTACCAGCTACAACATGCACTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGATG GGCGCCATCTATCCCGGCAACGGCGACACCTCCTACAACCAGAAATTCAAGGGCAGAGTGACCATGACCGAGGACA CCAGCACCGATACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAAGATACCGCCGTGTACTACTGTGCCACCTC CACCTACTATGGCGGCGACTGGTACTTCAACGTGTGGGGCCAGGGAACCACCGTGACAGTTTCT(SEQ ID NO.: 21)

>NH3-1

CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAA GGTGTCCGGCTACACCTTTACCAGCTACAACATGCACTGGGTCCGACAGACCCCTGGCAAAGGCCTTGAATGGATG GGCGCCATCTATCCCGGCAACGGCGACACCTCCTACAACCAGAAATTCAAGGGCAGAGTGACCATGACCGAGGACA CCAGCACCGATACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAAGATACCGCCGTGTACTACTGTGCCACCTC CACCTACTATGGCGGCGACTGGTACTTCAACGTGTGGGGCCAGGGAACCACCGTGACAGTTTCT(SEQ ID NO.: 22)

>NL2-1

GACATCCAGCTGACCCAGTCTCCAAGCTTTCTGAGCGCCAGCGTGGGCGACAGAGTGACCATTACATG TAGAGCCAGCAGCAGCGTGTCCTACATCCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTAC GCCACAAGCAATCTGGCCAGCGGCGTGCCAAGCAGATTTTCTGGCTCTGGCAGCGGCACCAGCTTCACCCTGACCA TATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTGGACCAGCAATCCTCCTACCTTTGGCGG AGGCACCAAGGTGGAAATCAAGCGGACAGTG(SEQ ID NO.:23)

>NL3

GAGATCGTGCTGACACAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAAGAGCCACACTGAGCTG TAGAGCCAGCAGCAGCGTGTCCTACATCCACTGGTATCAGCAGAAGCCCGGACAGGCCCCTAGACTGCTGATCTAC GCCACAAGCAATCTGGCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACAA TCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTGGACCAGCAATCCTCCTACCTTTGGCGG AGGCACCAAGGTGGAAATCAAGCGGACAGTG(SEQ ID NO.:24)

2, humanized antibody gene chemical synthesis and expression vector establishment

The heavy chain and light chain of the humanized antibody of above-mentioned design carry out gene chemical synthesis respectively, heavy chain be subcloned to PcDNA3.1-IgG1Fc expression vector, light chain are subcloned to pcDNA3.1-IgKc expression vector.Carrier is errorless through sequence verification Afterwards, endotoxin-free plasmid is prepared using the big pumping kit of Qiagen plasmid.

3, humanized antibody expression and purifying

3.1 take out LVTransm transfection reagent and single-chain antibody expression vector from refrigerator, after thaw at RT, use liquid-transfering gun Piping and druming mixes completely up and down.PBS or HBSS buffer is taken out, is warmed to room temperature.Take 2mL PBS to a hole of 6 orifice plates, difference 65 μ g pcDNA3.1-IgG1Fc and 65 μ g pcDNA3.1-IgKc are added, is blown and beaten above and below liquid-transfering gun after mixing well, is added 400 μ L LVTransm blows and beats mixing up and down with pipettor immediately, stands 10 minutes at room temperature.

3.2 are added to above-mentioned DNA/LVTransm compound in 50mL 293F-SVP16 cell, shake gently sufficiently mixed It is even.Cell is placed in 37 DEG C, 5%CO2 incubator, after 130RPM is cultivated 6~8 hours, the fresh FreeStyle of 50mL is added^TM 293Expression Medium culture medium, cell is placed back in and continues to cultivate in incubator.

After 3.3 continuous cultures 7 days, culture medium supernatant is collected by centrifugation, with 0.45 μm of membrane filtration, filtrate go to it is sterile from In heart pipe, single-chain antibody is purified using Protein A pillar.

4. the combination of streaming identification humanized antibody and target protein

4.1 from liquid nitrogen recovery Raji cell, use 1640,10%FBS complete medium to adjust cell state to logarithm Growth period.

Raji cell is divided into several pieces by 4.2, and the quantity of every part of cell is 1*10^6 cell, is resuspended using 1mL PBS Cell is separately added into the humanized whole antibodies of purifying, after mixing well, is incubated at room temperature half an hour.

4.3 800xg room temperatures are centrifuged 5 minutes, remove supernatant antibody-containing, are washed cell 3 times using PBS；

4.4 are added the Anti-human IgG of 2uL PE label, and after mixing well, room temperature, which is protected from light, is incubated for 30min；

4.5 800xg room temperatures are centrifuged 5 minutes, remove the supernatant containing secondary antibody, are washed cell 3 times using PBS；

4.6 are resuspended cell using 500uL PBS, carry out flow cytometer showed.

Experimental result

1. the heavy chain and light chain expression vector sequencing result of humanized antibody

The heavy chain and light chain expression vector of all buildings are sequenced through Sanger, sequencing result and the aim sequence (present invention Sequence) it is consistent.

2. humanized whole antibodies' FCM analysis result

As a result: as shown in Figure 1, according to flow cytometer detection as a result, choosing 2 flow cytometer detection parents from different humanized antibodies The table of a large amount of whole antibodies is carried out with the higher humanization carrier of power (Ruhab-NL2-1-NH2-1 and Ruhab-NL2-1-NH1-1) Up to purifying.

3, the expression and purification resisted entirely

As shown in Figure 2, the results showed that humanized antibody normal expression on protein level.

4, antibody humanization's context compares

As a result such as Fig. 3, shown in 4,5,6, heavy chain and light chain variable region for two humanized antibodies and source of mouse antibody are compared As a result.

5, antibody purification flow cytometer detection

Rumab antibody gradient dilution testing result is shown as shown in Figure 7.

Ruhab-NL2-1-NH1-1 antibody gradient dilution testing result is as shown in Figure 8.

Ruhab-NL2-1-NH2-1 antibody gradient dilution testing result is as shown in Figure 9.

As shown in Figure 10, MFI represents average fluorescent strength, the data obtained for preparation by streaming instrument.

Interpretation of result: according to flow cytometer detection result humanized antibody Ruhab-NL2-1-NH1-1 and chimeric antibody Rumab parent It is consistent with power, it can reach an order of magnitude.

The present invention has carried out humanization design and transformation to source of mouse CD20 antibody (Rituximab), and expresses corresponding Humanized antibody is verified through overflow-type verifying and affinity, be finally obtained 1 it is consistent with source of mouse affinity of antibody and reach one The humanized antibody sequence (NL2-1-NH1-1) of a order of magnitude.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Persongen Biomedicine (Suzhou) Co., Ltd.

<120>the humanization monoclonal antibody and its preparation of anti-CD20

<130> P2018-2420

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 1

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210> 2

<211> 7

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 2

Tyr Pro Gly Asn Gly Asp Thr

1 5

<210> 3

<211> 11

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 3

Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn

1 5 10

<210> 4

<211> 10

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 4

Arg Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 5

<211> 8

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 5

Ala Thr Ser Asn Leu Ala Ser Gly

1 5

<210> 6

<211> 9

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 6

Gln Gln Trp Thr Ser Asn Pro Pro Thr

1 5

<210> 7

<211> 121

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 7

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 8

<211> 109

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 8

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val

100 105

<210> 9

<211> 120

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 9

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Thr Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 10

<211> 109

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105

<210> 11

<211> 120

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 12

<211> 120

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 12

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Thr Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 13

<211> 119

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Asn

20 25 30

Met His Trp Val Arg Gln Thr Pro Gly Gln Gly Leu Glu Trp Met Gly

35 40 45

Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys

50 55 60

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met

65 70 75 80

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser

115

<210> 14

<211> 120

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 15

<211> 120

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 16

<211> 109

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105

<210> 17

<211> 360

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 17

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt ccgacagacc 120

cctggccaga gacttgaatg gatgggcgcc atctatcccg gcaacggcga cacctcctac 180

aaccagaaat tcaagggccg cgtgaccatc accagagaca catctgccag caccgcctac 240

atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagaagcacc 300

tactacggcg gcgactggta cttcaatgtg tggggccagg gcaccaccgt gacagtttct 360

<210> 18

<211> 360

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 18

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt ccgacagacc 120

cctggccaga gacttgaatg gatgggcgcc atctatcccg gcaacggcga cacctcctac 180

aaccagaaat tcaagggcag agtgaccatc accgccgaca catctgccag caccgcctac 240

atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagaagcacc 300

tactacggcg gcgactggta cttcaatgtg tggggccagg gcaccaccgt gacagtttct 360

<210> 19

<211> 357

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 19

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc tacaacatgc actgggtccg acagacccct 120

ggacagggac ttgaatggat gggcgccatc tatcccggca acggcgacac cagctacaac 180

cagaaattca agggccgcgt gaccatgacc agagacacca gcacctccac cgtgtacatg 240

gaactgagca gcctgagaag cgaggacacc gccgtgtact actgtgccag aagcacctac 300

tacggcggcg actggtactt caatgtgtgg ggccagggca ccaccgtgac agtttct 357

<210> 20

<211> 360

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 20

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt ccgacagaca 120

cctggacagg gactcgaatg gatgggcgcc atctatcccg gcaatggcga cacctcctac 180

aaccagaaat tcaagggcag agtgaccatg accgccgaca ccagcacaag caccgtgtac 240

atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagaagcacc 300

tactacggcg gcgactggta cttcaatgtg tggggccagg gcaccaccgt gacagtttct 360

<210> 21

<211> 360

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 21

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg tgtccggcta cacctttacc agctacaaca tgcactgggt ccgacaggcc 120

cctggcaaag gacttgaatg gatgggcgcc atctatcccg gcaacggcga cacctcctac 180

aaccagaaat tcaagggcag agtgaccatg accgaggaca ccagcaccga taccgcctac 240

atggaactga gcagcctgcg gagcgaagat accgccgtgt actactgtgc cacctccacc 300

tactatggcg gcgactggta cttcaacgtg tggggccagg gaaccaccgt gacagtttct 360

<210> 22

<211> 360

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 22

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60

tcctgcaagg tgtccggcta cacctttacc agctacaaca tgcactgggt ccgacagacc 120

cctggcaaag gccttgaatg gatgggcgcc atctatcccg gcaacggcga cacctcctac 180

aaccagaaat tcaagggcag agtgaccatg accgaggaca ccagcaccga taccgcctac 240

atggaactga gcagcctgcg gagcgaagat accgccgtgt actactgtgc cacctccacc 300

tactatggcg gcgactggta cttcaacgtg tggggccagg gaaccaccgt gacagtttct 360

<210> 23

<211> 327

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 23

gacatccagc tgacccagtc tccaagcttt ctgagcgcca gcgtgggcga cagagtgacc 60

attacatgta gagccagcag cagcgtgtcc tacatccact ggtatcagca gaagcccggc 120

aaggccccta agctgctgat ctacgccaca agcaatctgg ccagcggcgt gccaagcaga 180

ttttctggct ctggcagcgg caccagcttc accctgacca tatctagcct gcagcctgag 240

gacttcgcca cctactactg ccagcagtgg accagcaatc ctcctacctt tggcggaggc 300

accaaggtgg aaatcaagcg gacagtg 327

<210> 24

<211> 327

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 24

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcag cagcgtgtcc tacatccact ggtatcagca gaagcccgga 120

caggccccta gactgctgat ctacgccaca agcaatctgg ccagcggcat ccccgataga 180

ttttccggct ctggctccgg caccgacttc accctgacaa tcagcagact ggaacccgag 240

gacttcgccg tgtactactg ccagcagtgg accagcaatc ctcctacctt tggcggaggc 300

accaaggtgg aaatcaagcg gacagtg 327

Claims (10)

1. a kind of heavy chain variable region of antibody, which is characterized in that the antibody heavy chain variable region includes following three complementary decisions Area CDR:

CDR1 shown in SEQ ID NO.:1,

CDR2 shown in SEQ ID NO.:2, and

CDR3 shown in SEQ ID NO.:3,

Also, the affinity of heavy chain variable region shown in the affinity of the heavy chain variable region and the SEQ ID NO.:7 of source of mouse exists An order of magnitude.

2. a kind of heavy chain of antibody, which is characterized in that the heavy chain has heavy chain variable region as described in claim 1.

3. a kind of light chain variable region of antibody, which is characterized in that the antibody's light chain variable region includes following three complementary decisions Area CDR:

CDR1 shown in SEQ ID NO.:4,

CDR2 shown in SEQ ID NO.:5, and

CDR3 shown in SEQ ID NO.:6,

Also, the affinity of light chain variable region shown in the affinity of the light chain variable region and the SEQ ID NO.:8 of source of mouse exists An order of magnitude.

4. a kind of light chain of antibody, which is characterized in that the light chain has light chain variable region as claimed in claim 3.

5. a kind of antibody, which is characterized in that the antibody includes

(1) heavy chain variable region as described in claim 1；And/or

(2) light chain variable region as claimed in claim 3.

6. a kind of recombinant protein, which is characterized in that the recombinant protein includes

(i) heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；And

(ii) sequence label of optional assistance expression and/or purifying.

7. a kind of antibody preparation, which is characterized in that the antibody preparation includes:

(a) antibody as claimed in claim 5；And

(b) carrier, the carrier include: buffer, sterile water, optional surfactant.

8. a kind of kit, which is characterized in that the kit contains antibody preparation as claimed in claim 7, and contains The container of the antibody preparation.

9. a kind of CAR construction, which is characterized in that the scFv section of the antigen binding regions of the CAR construction is specificity It is incorporated into the combined area of CD20, and the scFv has heavy chain variable region as described in claim 1 and such as claim 3 institute The light chain variable region stated.

10. a kind of immunocyte of recombination, which is characterized in that the immunocyte expresses the as claimed in claim 9 of external source CAR construction.