CN109879966A

CN109879966A - Humanization design and expression verifying based on source of mouse CD19 antibody

Info

Publication number: CN109879966A
Application number: CN201811653085.8A
Authority: CN
Inventors: 杨林; 游凤涛
Original assignee: PERSONGEN BIOMEDICINE (SUZHOU) CO Ltd
Current assignee: PERSONGEN BIOMEDICINE (SUZHOU) CO Ltd
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2019-06-14
Anticipated expiration: 2038-12-29
Also published as: CN109879966B

Abstract

The present invention provides anti-CD19 Humanized monoclonal antibodies and its preparations.Specifically, the present invention provides a kind of new anti-CD19 humanized antibodies.Antibody of the invention can combine CD19 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 CD19.

Description

Humanization design and expression verifying based on source of mouse CD19 antibody

Technical field

The present invention relates to field of medicaments, more particularly to anti-CD19 Humanized monoclonal antibodies and its preparation.

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.Antibody humanization's technology can significantly reduce the immunogene of mouse monoclonal antibody Property.

Therefore, it is acted in all kinds of related diseases in view of CD19 and function, this field is suitable for treatment there is still a need for exploitation and suffers from The anti-CD19 humanized antibody of person.

Summary of the invention

The purpose of the present invention is carry out humanization design and expression, acquisition and mouse to source of mouse CD19 antibody (FMC63 clone) 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 CD19 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 light chain variable region of antibody, and the light chain variable region is selected from the group:

(1a) sequence light chain variable region as shown in SEQ ID NO.3；

(1b) by amino acid sequence shown in SEQ ID NO.3 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 (1a) described light chain variable region function the sequence as shown in SEQ ID NO.3 derived from light chain Variable region；

(2a) sequence light chain variable region as shown in SEQ ID NO.5；Or

(2b) by amino acid sequence shown in SEQ ID NO.5 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 (2a) described light chain variable region function the sequence as shown in SEQ ID NO.5 derived from light chain Variable region.

In another preferred example, there are selected from the group below corresponding to sequence shown in SEQ ID NO.1 for the light chain variable region Amino acid mutation: the 7th threonine (T), the 15th leucine (L), the 22nd serine (S), the 71st tyrosine (Y), 72nd serine (S), the 87th phenylalanine (F), the 104th leucine (L), or combinations thereof.

In another preferred example, the mutation that the light chain variable region corresponds to sequence shown in SEQ ID NO.1 is selected from the group: T7S, L15V, S22T, Y71F, S72T, F87Y, L104V, or combinations thereof.

In another preferred example, there are selected from the group below corresponding to sequence shown in SEQ ID NO.1 for the light chain variable region Amino acid mutation: the 1st asparatate (D), the 4th methionine (M), the 7th threonine (T), the 9th serine (S), 10 serines (S), the 17th asparatate (D), the 21st isoleucine (I), the 45th lysine (K), the 58th figured silk fabrics Propylhomoserin (V), the 71st tyrosine (Y), the 72nd serine (S), the 87th phenylalanine (F), or combinations thereof.

In another preferred example, the mutation that the light chain variable region corresponds to sequence shown in SEQ ID NO.1 is selected from the group: D1E, M4L, T7S, S9A, S10T, D17E, I21L, K45R, V58I, Y71F, S72T, F87Y, or combinations thereof.

In another preferred example, the light-chain variable sequence is as shown in SEQ ID NO.3 or 5.

The second aspect of the present invention, provides a kind of light chain of antibody, and the light chain has such as first aspect present invention The 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.

The third aspect of the present invention provides a kind of heavy chain variable region of antibody, and the heavy chain variable region is selected from the group:

(1c) sequence heavy chain variable region as shown in SEQ ID NO.4；

(1d) by amino acid sequence shown in SEQ ID NO.4 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 (1c) described heavy chain variable region function the sequence as shown in SEQ ID NO.4 derived from heavy chain Variable region；

(2c) sequence heavy chain variable region as shown in SEQ ID NO.6；Or

(2d) by amino acid sequence shown in SEQ ID NO.6 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 (2c) described heavy chain variable region function the sequence as shown in SEQ ID NO.6 derived from heavy chain Variable region.

In another preferred example, there are selected from the group below corresponding to sequence shown in SEQ ID NO.2 for the heavy chain variable region Amino acid mutation: the 17th serine (S), the 20th valine (V), the 48th leucine (L), the 67th leucine (L), 82nd methionine (M), the 85th leucine (L), the 92nd isoleucine (I), or combinations thereof.

In another preferred example, the mutation that the heavy chain variable region corresponds to sequence shown in SEQID NO.2 is selected from the group: S17T, V20L, L48I, L67V, M82L, L85V, I92V, or combinations thereof.

In another preferred example, there are selected from the group below corresponding to sequence shown in SEQ ID NO.2 for the heavy chain variable region Amino acid mutation: the 10th glycine (G), the 15th serine (S), the 17th serine (S), the 19th serine (S), 20 valines (V), the 44th glycine (G), the 49th glycine (G), the 85th leucine (L), the 115th serine (S), or combinations thereof.

In another preferred example, the mutation that the heavy chain variable region corresponds to sequence shown in SEQ ID NO.2 is selected from the group: G10A, S15T, S17T, S19T, V20L, G44A, G49A, L85M, S115T, or combinations thereof.

In another preferred example, the weight chain variabl area sequence is as shown in SEQ ID NO.4 or 6.

The fourth aspect of the present invention, provides a kind of heavy chain of antibody, and the heavy chain has such as third aspect present invention The 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.

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

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

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

Alternatively, the antibody includes light chain as described in respect of the second aspect of the invention；And/or such as fourth aspect present invention institute The heavy chain stated.

In another preferred example, the antibody has the light chain variable region as shown in SEQ ID NO.3 or 5；And/or such as Heavy chain variable region shown in SEQ ID NO.4 or 6.

In another preferred example, the antibody has mutation selected from the group below:

(a1) in another preferred example, the light chain variable region exists corresponding to sequence shown in SEQ ID NO.1 and is selected from down The amino acid mutation of group: the 7th threonine (T), the 15th leucine (L), the 22nd serine (S), the 71st tyrosine (Y), the 72nd serine (S), the 87th phenylalanine (F), the 104th leucine (L), or combinations thereof；And/or

(b1) there are amino acid mutations selected from the group below corresponding to sequence shown in SEQ ID NO.2 for the heavy chain variable region: 17th serine (S), the 20th valine (V), the 48th leucine (L), the 67th leucine (L), the 82nd egg ammonia Sour (M), the 85th leucine (L), the 92nd isoleucine (I), or combinations thereof.

(a2) there are amino acid mutations selected from the group below corresponding to sequence shown in SEQ ID NO.1 for the light chain variable region: 1st asparatate (D), the 4th methionine (M), the 7th threonine (T), the 9th serine (S), the 10th serine (S), the 17th asparatate (D), the 21st isoleucine (I), the 45th lysine (K), the 58th valine (V), 71 tyrosine (Y), the 72nd serine (S), the 87th phenylalanine (F), or combinations thereof；And/or

(b2) there are amino acid mutations selected from the group below corresponding to sequence shown in SEQ ID NO.2 for the heavy chain variable region: 10th glycine (G), the 15th serine (S), the 17th serine (S), the 19th serine (S), the 20th valine (V), the 44th glycine (G), the 49th glycine (G), the 85th leucine (L), the 115th serine (S) or its group It closes.

In another preferred example, the light-chain variable sequence of the antibody is as shown in SEQ ID NO.3 or 5；And/or it is described The weight chain variabl area sequence of antibody is as shown in SEQ ID NO.4 or 6.

In another preferred example, the light-chain variable sequence of the antibody is as shown in SEQ ID NO.3；And the antibody Weight chain variabl area sequence as shown in SEQ ID NO.4.

In another preferred example, the antibody has sequence light chain variable region as shown in SEQ ID NO.5；And it is described Antibody has sequence heavy chain variable region as shown in SEQ ID NO.6.

In another preferred example, the antibody is humanized antibody.

In another preferred example, the antibody is specific binding CD19.

In another preferred example, the KD value (M) of the affinity of the antibody on human CD19 is 2.0E-10~1.0E-8.

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.

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

(i) as described in the first aspect of the invention light chain variable region, light chain as described in respect of the second aspect of the invention, such as this hair Heavy chain variable region described in the bright third aspect, heavy 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.

The seventh aspect of the 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.

In another preferred example, in the preparation, the concentration of the antibody is 5-100mg/mL；Preferably 10- 70mg/mL, more preferably 20-60mg/mL.

In another preferred example, the buffer is selected from the group: citric acid buffer system, histidine buffer system or its Combination.

In another preferred example, the buffer is histidine buffer system.

In another preferred example, in the preparation, the concentration of the histidine buffer system is 0.5-20mM, preferably Ground is 1-10mM.

In another preferred example, histidine and histidine monohydrochloride are contained in the citric acid buffer system, is preferably contained 1-10mM histidine and 1-10mM histidine monohydrochloride, with the total weight of the antibody preparation.

In another preferred example, the buffer is citric acid buffer system.

In another preferred example, in the preparation, the concentration of the citric acid buffer system is 5-100mM, preferably Ground is 10-50mM.

In another preferred example, sodium citrate and sodium chloride are contained in the citric acid buffer system, preferably contains 10- 50mM sodium citrate and 50-200mM sodium chloride, with the total weight of the antibody preparation.

In another preferred example, the surfactant is selected from the group: Tween 80, polysorbas20, or combinations thereof.

In another preferred example, in the preparation, the surface-active contents are 0.005-0.2wt%, with The total weight of the antibody preparation.

In another preferred example, the surfactant is Tween 80, and the concentration is 0.02-0.1wt%.

In another preferred example, the preparation pH range is 5.0-7.5, preferably 5.5-7.

In another preferred example, the preparation is ejection preparation.

In another preferred example, the preparation includes: antibody described in fifth aspect present invention, histidine buffer body It is, the sterile water for injection of surfactant and surplus.

In another preferred example, the preparation includes:

Antibody 20-60mg/mL described in fifth aspect present invention；

1-10mM of histidine buffer system；

Tween 80 0.02-0.1wt%；

And the sterile water for injection as surplus, and the preparation pH range is preferred 5.5-6.5.

The eighth aspect of the present invention, provides a kind of kit, and the kit contains described in seventh aspect present invention Antibody preparation, and contain the container of the antibody preparation.

The ninth aspect of the present invention provides a kind of CAR construction, the antigen binding regions of the CAR construction ScFv sections are the combined area for specifically binding to CD19, and the scFv has light chain as described in the first aspect of the invention can Become area and heavy chain variable region as described in the third aspect of the present invention.

The tenth aspect of the present invention, provides a kind of immunocyte of recombination, the immunocyte expression external source as CAR construction described in ninth aspect present invention.

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).

The eleventh aspect of the present 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 twelveth 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 Light chain variable region described in invention first aspect, light chain as described in respect of the second aspect of the invention, as described in third aspect present invention Heavy chain variable region, antibody described in heavy chain as described in the fourth aspect of the present invention or fifth aspect present invention, such as present invention the Recombinant protein described in six aspects, the immunocyte as described in tenth aspect present invention, as described in tenth one side of the invention Antibody 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 CD19 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, kidney Tumour, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical carcinoma, oophoroma, lymph cancer, nose Pharynx cancer, adrenal tumor, tumor of bladder, non-small cell lung cancer (NSCLC), glioma, carcinoma of endometrium, or combinations thereof.

In another preferred example, the tumour is the tumour of high expression CD19.

In another preferred example, the drug or preparation are used to prepare prevention and/or treatment and CD19 (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 CD19 related disease.

In another preferred example, the detection reagent or kit are for CD19 albumen in test sample.

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

The thirteenth 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: light chain variable region as described in the first aspect of the invention, such as Light chain described in second aspect of the present invention, heavy chain variable region as described in the third aspect of the present invention, such as fourth aspect present invention institute Antibody described in the heavy 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.

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

In another preferred example, the pharmaceutical composition is injection.

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

In another preferred example, the tumour is the tumour of high expression CD19.

The fourteenth aspect of the present invention, provides a kind of polynucleotides, and the polynucleotide encoding is selected from the group below more Peptide:

(1) as described in the first aspect of the invention light chain variable region, light chain as described in respect of the second aspect of the invention, such as this hair Described in heavy chain variable region described in the bright third aspect, heavy 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 such as fourteenth aspect of the present invention institute The polynucleotides stated.

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 sixteenth aspect of the present invention, provides a kind of genetically engineered host cell, and the host cell contains Integration is just like the polynucleotides as described in fourteenth aspect of the present invention in carrier or genome as described in the fifteenth aspect of the present invention.

The seventeenth aspect of the present invention provides CD19 in a kind of vitro detection (including diagnostic or nondiagnostic) sample The method of albumen, the method includes the steps:

(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 CD19 eggs in sample It is white.

The eighteenth aspect of the present invention, provides a kind of detection plate, and the detection plate includes: substrate (support plate) and surveys Strip, the test-strips contain the antibody as described in fifth aspect present invention or the antibody as described in the tenth one side of the invention Drug conjugates.

The nineteenth aspect of the present invention provides a kind of kit, includes: in the kit

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

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

Alternatively, the kit contain the present invention the 18th in terms of as described in detection plate.

The twentieth 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.

On the one hand of the invention the 20th, provides the side of a kind for the treatment of and CD19 expression or dysfunction related disease Method, which comprises the antibody drug of antibody as described in fifth aspect present invention, the antibody is applied to the object of needs The CAR-T cell of conjugate or the expression antibody, or combinations thereof.

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 the vector construction figure of single-chain antibody expression.

Fig. 2 shows single-chain antibody FCM analysis result.

Fig. 3 shows the electrophoretogram of entirely anti-expression and purification.

Fig. 4 shows that antibody humanization's context compares, wherein (A) is clone T1L1H4, (B) is clone T4L2H2.

Fig. 5 shows the selection result of coupling buffer.

Fig. 6 shows the result of the final coupling amount of ligand.

Fig. 7 shows different monoclonal antibodies and the kinetic constant measurement result with CD19, wherein (A) and (B) indicates inosculating antibody Body (chimeric)；(C) and (D) indicates T1L1H4；(E) and (F) indicates T4L2H2.

Specific embodiment

The present inventor is by extensive and in-depth research, by largely screening, unexpectedly obtains a kind of excellent with affinity The anti-CD19 humanized antibody of different and good structural stability.Specifically, the present invention is simultaneous when selecting humanization skeleton area Similitude and human body frequency of use are cared for, is selected human antibody template T1-T4 (such as two groups of sequences of T1H4, T1L1, T4H2, T4L2) Skeleton area carry out humanization, and to the humanized antibody of acquisition carry out screen mutation.Antibody after humanization, is especially mutated Humanized antibody afterwards reach with affinity similar in chimeric antibody, by the humanized antibody solubility and endogenous fluorescence Primary Study, it was demonstrated that the humanization has had preliminary druggability, and there is the humanization list for being developed further into targeted therapy in future Antiradiation drug.And preliminary patent medicine Journal of Sex Research is carried out to the antibody after humanization, screened and the anti-CD19 humanized antibody has been determined Pharmaceutical formulation.Specifically, the present invention has carried out humanization design and transformation to source of mouse CD19 antibody (FMC63 clone), and Corresponding humanized antibody is expressed, is verified through overflow-type verifying and affinity, is finally obtained 2 and source of mouse affinity of antibody Consistent humanized antibody sequence (T1L1H4, T4L2H2).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 19 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 19 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%.

CD19

CD19 molecule is the treatment very promising target spot of B system tumour, and CD19 is on most of B cell malignant cell surface CD19 molecule is all expressed, and the cell of normal cell surface expression CD19 molecule is only limited to B cell and follicularis Dendritic Cells, More importantly CD19 molecule is not expressed on pluripotential hemopoietic stem cell surface.

Source of mouse FMC63 variable region sequences information

FMC63 mouse monoclonal antibody light chain variable region (SEQ ID NO.1)

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

FMC63 mouse monoclonal antibody heavy chain variable region (SEQ ID NO.2)

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

Humanization T1L1H4 variable region sequences information

Humanization monoclonal antibody T1L1H4 light chain variable region (SEQ ID NO.3)

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGT DFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKVEI

Humanization monoclonal antibody T1L1H4 heavy chain variable region (SEQ ID NO.4)

QVQLQESGPGLVAPSETLSLTCTVSGVSLPDYGVSWIRQPPRKGLEWIGVIWGSETTYYNSALKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

Humanization T4L2H2 variable region sequences information

Humanization monoclonal antibody T4L2H2 light chain variable region (SEQ ID NO.5)

EIVLTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPDRFSGSGSGT DFTLTISRLEPEDIAVYYCQQGNTLPYTFGQGTKLEI

Humanization monoclonal antibody T4L2H2 heavy chain variable region (SEQ ID NO.6)

QVTLRESGPALVKPTQTLTLTCTFSGVSLPDYGVSWIRQPPGKALEWLAVIWGSETTYYNSALKSRLTI SKDTSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGTTVTVSS

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-CD19 prepared according to this field knowledge and skills in the present invention Body.With CD19 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 CD19 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, CD19).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 CD19 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 CD19 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 CD19 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 CD19。

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

Anti- CD19 humanized antibody

The present invention provides anti-CD19 humanized antibody (hereinafter referred to as CD19 antibody).Specifically, the present invention provides a kind of needle The humanized antibody of high specific and high-affinity to CD19 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, IGKV3-20*01 and IGHV1-69*01 two have been selected The skeleton area of sequence carries out humanization.The present invention selects human antibody Germline skeleton area to carry out CDR transplanting, the people constructed in this way Source antibody structure is more stable, expression quantity is high, immunogenicity is low, druggability is higher.

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

FMC63 humanized antibody light chain variable region

DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKVEI(SEQ ID NO.:3)

FMC63 humanised antibody heavy chain variable region

QVQLQESGPGLVAPSETLSLTCTVSGVSLPDYGVSWIRQPPRKGLEWIGVIWGSETTYYNSALKSRVT ISVDTSKNQFSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS(SEQ ID NO.:4)

FMC63 humanized antibody light chain variable region

EIVLTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPDRFSGSGSG TDFTLTISRLEPEDIAVYYCQQGNTLPYTFGQGTKLEI(SEQ ID NO.5)

FMC63 humanised antibody heavy chain variable region

QVTLRESGPALVKPTQTLTLTCTFSGVSLPDYGVSWIRQPPGKALEWLAVIWGSETTYYNSALKSRLT ISKDTSKNQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGTTVTVSS(SEQ ID NO.6)

In another preferred example, the constant region of the humanized antibody is the same as the constant of anti-CD19 mouse monoclonal antibody FMC63 Area is identical.

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 CD19, 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 CD19 mouse monoclonal antibody humanization modified, and the antibody after humanization reaches and chimeric antibody phase Close affinity passes through the Primary Study to the humanized antibody solubility and endogenous fluorescence, it was demonstrated that the humanization has had just There are the humanization monoclonal antibody medicine for being developed further into targeted therapy in the druggability of step, future.

The preparation of antibody

Any method for being suitable for generating monoclonal antibody can be used in generating CD19 antibody of the invention.For example, can use Animal is immunized in connection or naturally occurring CD19 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 CD19 of any suitable form all can serve as immunogene (antigen), special to CD19 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 CD19 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 CD19 protein molecular, thus can be used for preventing and treating CD19 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) present invention develops a kind of CD19 antibody of humanization.

(2) present invention obtains a kind of CD19 antibody sequences of humanization.

(3) present invention can reduce the immunological rejection that source of mouse CD19 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 (FMC63 clone) of CD19 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 Results simulate as a result, choosing It selects heavy chain and light chain and selects four different human antibody template T1-T4 respectively, and the framework region after humanization is returned Multiple mutation (back mutation), designs candidate humanized antibody sequence.Particular sequence information is shown in Table 3 and table 4.

2, humanized antibody gene chemical synthesis and expression vector establishment

The Humanized single chain antibody segment of above-mentioned design is subjected to gene chemical synthesis respectively, as template, carries out overlap PCR, assembles the single-chain antibody of VL- (G4S) 3-VH, i.e. the heavy chain of T1-T4 template and light chain carries out combination of two respectively, will combine Single-chain antibody afterwards is subcloned using restriction enzyme site EcoRI-BglII into single-chain antibody expression vector pFUSE-hIgG1-Fc2 (carrier schematic diagram such as Fig. 1, Fc sequence behaviour IgG1Fc).Carrier uses the big odd test of Qiagen plasmid after sequence verification is errorless Agent box prepares endotoxin-free 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 130 μ g pFUSE-hIgG1-Fc2 are added, is blown and beaten above and below liquid-transfering gun after mixing well, 400 μ L LVTransm is added, use immediately Pipettor blows and beats mixing up and down, 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%CO₂After 130RPM is cultivated 6~8 hours, the fresh CD-CHO culture of 50mL is added in incubator Cell is placed back in and continues to cultivate in incubator by base.

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, recovery K562 ghost and K562-CD19 recombinant cell strain from liquid nitrogen, it is complete using 1640,10%FBS Culture medium adjusts cell state to logarithmic growth phase.

4.2, two kinds of cells are respectively divided into several pieces, the quantity of every part of cell is 1*10^6 cell, uses 1mL PBS Cell is resuspended, is separately added into the Humanized single chain antibody of purifying, each single-chain antibody is both needed to K562 and K562-CD19 with blank It is incubated for, after mixing well, is incubated at room temperature half an hour.

4.3,800xg room temperature is centrifuged 5 minutes, removes supernatant antibody-containing, is washed cell 3 times using PBS；

4.4, the anti-human igg (Anti-human IgG) of 2uL PE label is added, after mixing well, room temperature is protected from light incubation 30min；

4.5,800xg room temperature is centrifuged 5 minutes, removes the supernatant containing secondary antibody, is washed cell 3 times using PBS；

4.6, cell is resuspended using 500uL PBS, carries out flow cytometer showed.

Two, experimental result

1, single-chain antibody expression vector sequencing result

The single-chain antibody expression vector of all buildings is sequenced through Sanger, except clone T1L2H1 is in humanization framework region Occur except a point mutation in FR1, all antibody expression vectors are completely correct.

2, single-chain antibody FCM analysis result

As a result: according to flow cytometer detection as a result, from different humanized antibody templates choose 2 flow cytometer detection affinity compared with High humanization carrier (T1L1H4 and T4L2H2) carries out the building and intact antibody purifying of complete anti-carrier, for affine Power detection.As a result see Fig. 2.

3, the expression and purification resisted entirely

The result characterization of anti-expression and purification is shown in Fig. 3 entirely.

In Fig. 3, swimming lane 1 represents the protein electrophoresis band of chimeric antibody, and swimming lane 2 represents the albumen of humanization T1L1H4 antibody Electrophoretic band, swimming lane 3 represent the protein electrophoresis band of humanization T4L2H2 antibody.

4, antibody humanization's context compares

Antibody humanization's context comparing result is shown in Fig. 4.

In Fig. 4, A figure refers to the T1L1H4 and the heavy chain of source of mouse FMC63 and the comparison result of light chain variable region of humanization；B Figure refers to the T4L2H2 and the heavy chain of source of mouse FMC63 and the comparison result of light chain variable region of humanization.

5, Biacore affinity determination

5.1 ligand coupling preenrichments experiment

Experiment condition:

Ligand: CD19；

Test pH:10mM Acetate 5.5/5.0/4.5/4.0；

Flow velocity: 10 μ l/min；

Buffer: HBS-EP+buffer；

Selection result is shown in Fig. 5, selects Acetate pH 5.0 as coupling buffer.

The experiment of 5.2 ligand couplings

Experiment condition:

Ligand: CD19；

Coupling buffer: 10mM Acetate 5.0；

Target coupling amount: 1000RU；

As a result see Fig. 6, final coupling amount is 1035.8RU.

5.3HM1711091-chimeric, the combination of HM1711091-T1L1H4, HM1711091-T4L2H2 and CD19 surveys It is fixed

Experiment condition:

Flow velocity: 30 μ l/min；

Binding time: 180s；

Dissociation time: 180s；

Regenerative agent: Glycine 1.5,30s；

1) it is fitted into (chimeric): 3.125/6.25/12.5/25/50/100ug/ml

As a result see Fig. 7 A and Fig. 7 B.

The kinetic constant of HM1711091-chimeric and CD19 is as follows:

Association rate constant ka=1.725 × 10⁵M-1s-1；

Dissociation rate constant kd=0.004416s-1；

Dissociation equilibrium constant KD=2.560 × 10^-8M。

2)T1L1H4:3.125/6.25/12.5/25/50/100ug/ml

As a result see Fig. 7 C and Fig. 7 D.

The kinetic constant of HM1711091-T1L1H4 and CD19 is as follows:

Association rate constant ka=2.779 × 10⁵M-1s-1；

Dissociation rate constant kd=0.004406s-1；

Dissociation equilibrium constant KD=1.586 × 10^-8M。

3)T4L2H2:3.125/6.25/12.5/25/50/100ug/ml

As a result see Fig. 7 E and Fig. 7 F.

The kinetic constant of HM1711091-T4L2H2 and CD19 is as follows:

Association rate constant ka=9.855 × 10⁴M-1s-1；

Dissociation rate constant kd=0.002799s-1；

Dissociation equilibrium constant KD=2.840 × 10^-8M。

The binding constant experimental result of three kinds of antibody and CD19 are shown in Table 2.

The binding constant of table 2 three kinds of antibody and CD19

The result shows that: according to affinity testing result (Fig. 7 and table 2), humanized antibody T1L1H4, T4L2H2 with it is chimeric (Chimeric) Dissociation equilibrium constant KD is in the same order of magnitude, illustrates the affinity one of humanized antibody and chimeric antibody It causes.

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.

3 humanization candidate antibodies amino acid sequence information of table

4 humanization candidate antibodies nucleic acid coding sequence information of table

Sequence table

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

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 6

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 6

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 7

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 8

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 9

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 11

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 12

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 12

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Leu Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 13

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 14

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 14

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 15

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 15

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 16

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 16

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 17

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 17

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 18

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 19

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 20

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 Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 21

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 21

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 Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 22

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 22

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 Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105

<210> 23

<211> 120

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 23

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 106

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 24

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 Gln Asp Ile Ser Lys Tyr

20 25 30

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

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105

<210> 25

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 25

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 26

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 26

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 27

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 27

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagcgaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat ccggcagcct 120

cctagaaaag gcctggaatg gatcggcgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 28

<211> 371

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 28

caggttcagc tgcaagagtc tggacctgga ctggtggccc ctagcgaaac actgagcctg 60

acctgtacag ttagcggcgt gtccctgcct gactatggcg tgtcctggat cagacagcct 120

cctcggaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

agctggaaat c 371

<210> 29

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 29

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccttca ccataagcag cctgcagcct 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 30

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 30

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccttta caatcagcag cctgcagcaa 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 31

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 31

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagcct 120

ggcggcgctc ccaagctgct gatctaccac acaagcagac tgcacagcgg cgtgcccagc 180

agattttctg gctctggcag cggcaccgac ttcaccttta caatcagcag cctgcagcaa 240

gaggatatcg ccacctacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 32

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 32

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcac 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccctggt caccatctcc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 33

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 33

caggttcagc tgcaagagtc tggccctggc ctggtcaagc ctagccaaac actgagcctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcac 120

cctggcaaag gcctggaatg gatcggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtccagagt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgacagc cgccgataca gccgtgtact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 34

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 34

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaaaccc 120

ggcaaggtgc ccaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 35

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 35

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaagaccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 36

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 36

gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60

atcacctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggcaagaccc ctaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180

agattttctg gcagcggctc tggcaccgac ttcaccctga caatcagcag cctgcagcaa 240

gaggacgtgg ccacctacta ttgccagcag ggcaacaccc tgccttacac ctttggccag 300

ggcaccaagc tggaaatc 318

<210> 37

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 37

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 38

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 38

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 39

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 39

caagtgaccc tgaaagaaag cggccctgtg ctggtcaagc ccaccgaaac actgaccctg 60

acctgtaccg tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggatacca gcaagagcca ggtggtgctg 240

accatgaaca acatggaccc cgtggacacc gccacctact actgtgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360

<210> 40

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 40

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggacttcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 41

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 41

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 42

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 42

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatcagcag gctggaacaa 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggcgga 300

ggcaccaagg tggaaatc 318

<210> 43

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 43

caagtgaccc tgagagagtc tggccctgct ctggtcaagc ccacacagac cctgacactg 60

acctgcacct tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggacacca gcaagaacca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccag acactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 44

<211> 360

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 44

caagtgaccc tgagagagtc tggccctgct ctggtcaagc ccacacagac cctgacactg 60

acctgcacct tcagcggagt gtccctgcct gattatggcg tgtcctggat cagacagcct 120

cctggcaaag ccctggaatg gctggctgtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcagc aaggacacca gcaagaacca ggtggtgctg 240

accatgacca acatggaccc tgtggacacc gccacctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggattattgg ggccagggca ccaccgtgac cgtttcttct 360

<210> 45

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 45

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggacttcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggccag 300

ggcaccaagc tggaaatc 318

<210> 46

<211> 318

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 46

gagatcgtgc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca 60

ctgagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

ggacaggccc ctagactgct gatctaccac accagcagac tgcacagcgg catccccgat 180

agattttctg gcagcggctc cggcaccgac ttcaccctga caatctccag actggaaccc 240

gaggatatcg ccgtgtacta ctgccagcag ggcaacaccc tgccttacac atttggccag 300

ggcaccaagc tggaaatc 318

Claims

1. a kind of light chain variable region of antibody, which is characterized in that the light chain variable region is selected from the group:

(1a) sequence light chain variable region as shown in SEQ ID NO.3；

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

(2a) sequence light chain variable region as shown in SEQ ID NO.5；Or

(2b) by amino acid sequence shown in SEQ ID NO.5 by least one (such as 1-20, preferably 1-15, more preferably Ground 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) substitution of amino acid residue, missing, modification and/or Addition and formed, with (2a) described light chain variable region function the sequence as shown in SEQ ID NO.5 derived from light chain variable Area.

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

3. a kind of heavy chain variable region of antibody, which is characterized in that the heavy chain variable region is selected from the group:

(1c) sequence heavy chain variable region as shown in SEQ ID NO.4；

(1d) by amino acid sequence shown in SEQ ID NO.4 by least one (such as 1-20, preferably 1-15, more preferably Ground 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) substitution of amino acid residue, missing, modification and/or Addition and formed, with (1c) described heavy chain variable region function the sequence as shown in SEQ ID NO.4 derived from weight chain variable Area；

(2c) sequence heavy chain variable region as shown in SEQ ID NO.6；Or

(2d) by amino acid sequence shown in SEQ ID NO.6 by least one (such as 1-20, preferably 1-15, more preferably Ground 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) substitution of amino acid residue, missing, modification and/or Addition and formed, with (2c) described heavy chain variable region function the sequence as shown in SEQ ID NO.6 derived from weight chain variable Area.

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

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

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

(2) heavy chain variable region as claimed in claim 3；

Alternatively, the antibody includes light chain as claimed in claim 2；And/or heavy chain as claimed in claim 4.

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

(i) light chain variable region as described in claim 1, light chain as claimed in claim 2, weight as claimed in claim 3 Chain variable region, heavy 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 CD19, and the scFv has light chain variable region as described in claim 1 and such as claim 3 institute The heavy chain variable region stated.

10. a kind of antibody drug conjugates, which is characterized in that the antibody drug conjugates contain:

(a) antibody moiety, the antibody moiety are selected from the group: light chain variable region, such as claim 2 as described in claim 1 The light chain, heavy chain variable region as claimed in claim 3, heavy chain as claimed in claim 4 or such as claim 5 institute The antibody stated, or combinations thereof；With

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