CN107880128A

CN107880128A - A kind of anti-CD19 human antibody or antibody fragment and its methods and applications

Info

Publication number: CN107880128A
Application number: CN201711395512.2A
Authority: CN
Inventors: 杨光; 闵晨雨
Original assignee: Changzhou Velox Pharmaceutical Science & Technology Co Ltd
Current assignee: Changzhou Velox Pharmaceutical Science & Technology Co Ltd
Priority date: 2017-12-21
Filing date: 2017-12-21
Publication date: 2018-04-06
Anticipated expiration: 2037-12-21
Also published as: CN107880128B

Abstract

The invention discloses the human antibody of anti-CD19 a kind of or antibody fragment and its methods and applications, the antibody or antibody fragment include：Heavy chain and light chain；Heavy chain and light chain include variable region, and variable region includes complementary determining region.Complementary determining region CDR1, CDR2 and CDR3 of heavy chain are represented with HCDR1, HCDR2 and HCDR3 respectively；Complementary determining region CDR1, CDR2 and CDR3 of light chain are represented with LCDR1, LCDR2 and LCDR3 respectively；HCDR1 amino acid sequence includes SEQ ID NO：3；HCDR2 amino acid sequence includes SEQ ID NO：4；HCDR3 amino acid sequence includes SEQ ID NO：5；LCDR1 amino acid sequence includes SEQ ID NO：6；LCDR2 amino acid sequence includes DVS；LCDR3 amino acid sequence includes SEQ ID NO：7.The antibody or antibody fragment of the present invention be capable of high specific with people source CD19 protein bindings, expression is engineered in T cell using Chimeric antigen receptor T cell technology to integrate, obtained Chimeric antigen receptor T cell can be used in treating the hematologic cancer related to CD19 expression.

Description

A kind of anti-CD19 human antibody or antibody fragment and its methods and applications

Technical field

The invention belongs to the biological therapy technical field of malignant tumour, and it is related to a kind of anti-CD19 antibody, and in particular to A kind of anti-CD19 human antibody or antibody fragment and its methods and applications.

Background technology

CD19 is expressed on B systems cell (not including mature plasme cell) and dendritic cells,follicular, is a kind of important letter Number transduction molecule, adjusts growth activation and the activation of bone-marrow-derived lymphocyte, regulation bone-marrow-derived lymphocyte antigen receptor or other surfaces by Played an important role in the signal threshold value of body, be that the important membrane relevant with bone-marrow-derived lymphocyte differentiation, activation, propagation and antibody generation resists Original, it is the preferably mark for diagnosing bone-marrow-derived lymphocyte system's tumour and identifying bone-marrow-derived lymphocyte.

Many pernicious patients of B cell can not be cured using standard treatment.In addition, traditional treatment option usually have it is serious Side effect.Trial was carried out in cancer immunotherapy, but several obstacles extremely to be difficult to obtain Clinical efficacy. Although having identified hundreds of so-called tumour antigens, these antigens are typically derived from itself and therefore immunogenicity It is weak.In addition, tumour can resist the starting of immune attack and expansion by several mechanism.

Knubble biological immunization therapy is the 4th kind of means of the oncotherapy after surgical operation, chemotherapy and radiation.It is chimeric Antigen receptor (chimeric antigen receptor, with CAR-T cell technologies) is the tumour for obtaining important breakthrough in recent years Biological immune treats new tool.CAR-T cell technologies be by can specific recognition tumour antigen engineering carrier import people In body immunocyte (such as lethal CDS+T cells), this group of T cells are made to encode out tumour specific antigen, and then identify tumour The receptor target killing tumor cell of cell surface.

Compared with conventional cellular immunotherapy means, CAR-T cell technologies have following advantage：First, with CAR-T Identification of the cell to antigen is processed independent of antigen and the antigen presentation of HLA molecules, the tumour cell of immunologic escape often have The characteristics of low expression HLA and Proteasome antigen are processed, also can be by with CAR-T cell recognitions, therefore CAR-T cells against tumor is thin Born of the same parents have more preferable targeting and lethal；Second, CAR-T cell can not only identify the albumen of cell surface, can also identify thin The space structure such as the carbochain of cellular surface and sugar chain, there is the characteristics of broadly tumor cell；3rd, thin with CAR-T Cellular surface also has the expression of costimulatory molecules except the expression of specific antibody, the expression of costimulatory molecules and cell factor Survival and the vigor of T cell can be effectively facilitated by adding, and clinically improve the persistence of immunization therapy to a greater extent.

A series of current animal experiments and clinical test all demonstrate CAR-T cells to neoplastic hematologic disorder and treatment of solid tumors Validity and security, CAR-T cells have huge application potential and development prospect in the clinical treatment of tumour, are futures Cure the sharp weapon of tumour.

It is in progress recently using the therapy of Chimeric antigen receptor (CAR) modification Autologous T cells (CAR-T) rapid, utilizes human body Self immune system shows very big hope in terms for the treatment of malignant B cell tumor disease and other cancers.

The content of the invention

It is an object of the invention to provide the human antibody of anti-CD19 a kind of or antibody fragment and its methods and applications, and this is anti- Body or antibody fragment solve the problems such as targeting difference of existing cellular immunotherapy, be capable of high specific with people source CD19 eggs It is white to combine, be engineered using Chimeric antigen receptor T cell technology (CAR-T technologies) in T cell express be incorporated into it is in CAR, With reference to CD19 human antibody fragments, obtained Chimeric antigen receptor T cell can be used in treating the blood related to CD19 expression Liquid cancer.

In order to achieve the above object, the invention provides a kind of anti-CD19 human antibody or antibody fragment, the antibody Or antibody fragment includes：Heavy chain and light chain；Described heavy chain and light chain include variable region, and described variable region is determined including complementation Determine area CDR1, CDR2 and CDR3；Complementary determining region CDR1, CDR2 and CDR3 of described heavy chain respectively with HCDR1, HCDR2 and HCDR3 is represented；Complementary determining region CDR1, CDR2 and CDR3 of described light chain are represented with LCDR1, LCDR2 and LCDR3 respectively.

Wherein, described HCDR1 amino acid sequence includes SEQ ID NO：3 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：3.

Wherein, described HCDR2 amino acid sequence includes SEQ ID NO：4 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：4.

Wherein, described HCDR3 amino acid sequence includes SEQ ID NO：5 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：5.

Wherein, described LCDR1 amino acid sequence includes SEQ ID NO：6 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：6.

Wherein, described LCDR2 amino acid sequence includes DVS, i.e. sequence Asp Val Ser.

Wherein, described LCDR3 amino acid sequence includes SEQ ID NO：7 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：7.

Described heavy chain variable amino acid sequence includes SEQ ID NO：1 or the sequence in one or a few amino The SEQ ID NO of acid mutation：1；

Described chain variable region amino acid sequence includes SEQ ID NO：2 or the sequence in one or a few amino The SEQ ID NO of acid mutation：2.

Described heavy chain represents that described light chain is represented with VL with VH；Described antibody or the amino acid sequence of antibody fragment Row also include VH-GGGGGS-VL and/or VL-GGGGGS-VH.

Described antibody or antibody fragment are with 2 × 10^-6M or lower KD values and people's CD19 protein bindings.

Present invention also offers a kind of nucleotides, the nucleotide coding anti-CD19 as mentioned human antibody or antibody Fragment；Described nucleotides includes：For encoding the nucleotide sequence SEQ ID NO of described weight chain variable district：8, Yi Jiyong In the nucleotide sequence SEQ ID NO of the described light chain variable district of coding：9.

Present invention also offers a kind of Chimeric antigen receptor, this receptor contain anti-CD19 human antibody as mentioned or Antibody fragment.

This receptor includes what is be sequentially connected：Signal peptide sequence, CD19 binding structural domains, detection label, hinge area and cross-film Domain, and functional signal conducting structure domain.

Wherein, described CD19 binding structural domains are scFv, and the scFv includes the human antibody of anti-CD19 as mentioned Or antibody fragment.

Wherein, described hinge area and membrane spaning domain include：Hinge area Hinge TM and transmembrane structure CD8, the hinge Area Hinge TM and transmembrane structure CD8 amino acid sequence include SEQ ID NO：10 or the sequence in one or a few ammonia The SEQ ID NO of base acid mutation：10；

Described functional signal conducting structure domain includes：CD28,4-1BB and the CD3Zeta being sequentially connected；

Described CD28 amino acid sequence includes SEQ ID NO：11 or the sequence in one or a few amino acid The SEQ ID NO of mutation：11；

Described 4-1BB amino acid sequence includes SEQ ID NO：12 or the sequence in one or a few amino acid The SEQ ID NO of mutation：12；

Described CD3Zeta amino acid sequence includes SEQ ID NO：13 or the sequence in one or a few amino The SEQ ID NO of acid mutation：13；

Described signal peptide sequence includes CSF2RA, and its amino acid sequence includes SEQ ID NO：14 or the sequence in one Or the SEQ ID NO of a few amino acid mutation：14；

Described detection label includes C-Myc, and its amino acid sequence includes SEQ ID NO：15 or the sequence in one or The SEQ ID NO of a few amino acid mutation：15.

Preferably, described VH-GGGGGS-VL includes following sequence：

MAQVQLVETGGGLVKPRGSLRLSCAASGFSFSDYYMTWIRKAPGRGLEWIANIRERRGNTRYAESVKGRFTIARDNA MNSVYLQMNSLRVEDTGIYYCARSHRSGWFLDVWGQGTTVTVSSGGGGGSQSALTQPASVSGSPGQSITISCTGTSG DVGAYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSRNTASLTISGLQAEDEADYYCSSYTSSSTPLFGGG TKLTVLG。

Preferably, described VL-GGGGGS-VH includes following sequence：

QSALTQPASVSGSPGQSITISCTGTSGDVGAYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSRNTASLTI SGLQAEDEADYYCSSYTSSSTPLFGGGTKLTVLGGGGGGSMAQVQLVETGGGLVKPRGSLRLSCAASGFSFSDYYMT WIRKAPGRGLEWIANIRERRGNTRYAESVKGRFTIARDNAMNSVYLQMNSLRVEDTGIYYCARSHRSGWFLDVWGQG TTVTVSS。

Present invention also offers a kind of T cell for being used to treat or prevent tumour, the T cell contains inosculating antibody as mentioned Original receptor.

Present invention also offers a kind of method for preparing Chimeric antigen receptor, this method is contained as described using transposons structure Chimeric antigen receptor T cell.

Present invention also offers a kind of method for detecting effector cell and being activated, this method includes：The swollen of CD19 will be expressed Oncocyte and do not express CD19 tumour cell and be used as target cell, be expressed as expressing CD19 groups and do not express CD19 groups, Chimeric antigen receptor CD19-C8scFv-CAR-T cells will be expressed as effector cell, CD19 groups will be expressed and do not express CD19 Group co-cultures with effector cell respectively, detects cell factor IFN-γ, IL-2, TNF-α and GM-CSF level；If expressing The level for the cell factor that CD19-C8scFv-CAR-T cells are secreted, which is higher than, in CD19 groups does not express CD19 groups, described CAR-T Effector cell is activated.

Wherein, described CD19-C8scFv-CAR-T cells are the T cell for being used to treat or prevent tumour as mentioned.

The anti-CD19 of present invention human antibody or antibody fragment and its methods and applications, solves existing cellular immunity The problems such as targeting difference for the treatment of, there is advantages below：

(1) of the invention antibody or antibody fragment be capable of high specific with people source CD19 protein bindings, utilize inosculating antibody Original receptor T cell technology (CAR-T technologies) is engineered expression in T cell and is incorporated into human antibody in CAR, with reference to CD19 Fragment, obtained Chimeric antigen receptor T cell can be used in treating the hematologic cancer related to CD19 expression；

(2) Chimeric antigen receptor T cell of the invention being capable of targeted malignant B cell tumor disease cell surface mark point Son；

(3) present invention construct expression after can specific recognition B cell malignant tumour surface C D19 molecules CD19- C8scFv-CAR transposons, after transfecting T cells, it specific can identify B cell malignant tumour relevant cell and make its apoptosis, So as to reach healing purpose.

Brief description of the drawings

Fig. 1 is the bar chart that the single-stranded scFv antibody fragments results of CD19 are screened in experimental example 1.

Fig. 2 is the full people source scFv antibody of anti-CD19 and CD19 binding kineticses curve maps.

Fig. 3 is the structure of the CD19-C8scFv-CAR piggyBac transposon expression vectors used in the present invention Figure.

Fig. 4 is the flow cytometer detection of CD19-C8scFv-CAR T cells surface C AR expression rates made from experimental example 4 of the present invention Result figure.

Fig. 5 divides thin after being co-cultured with target cell for obtained CD19-C8scFv-CAR T cells in the embodiment of the present invention 4 The secretory volume column diagram of intracellular cytokine IFN-γ.

Fig. 6 divides thin after being co-cultured with target cell for obtained CD19-C8scFv-CAR T cells in the embodiment of the present invention 4 Intracellular cytokine IL-2 secretory volume column diagram.

Fig. 7 divides thin after being co-cultured with target cell for obtained CD19-C8scFv-CAR T cells in the embodiment of the present invention 4 The secretory volume column diagram of intracellular cytokine TNF-α.

Fig. 8 divides thin after being co-cultured with target cell for obtained CD19-C8scFv-CAR T cells in the embodiment of the present invention 4 Intracellular cytokine GM-CSF secretory volume column diagram.

Embodiment

Term defines

" antibody " refers to comprising at least two weight (H) chains and two light (L) chain being mutually connected to each other by disulfide bond Glycoprotein, or its antigen-binding portion thereof.Antibody includes complete antibody and its any antigen-binding fragment or single-chain antibody.

" heavy chain " is made up of weight chain variable district (VH) and heavy chain constant region.

" heavy chain constant region " is made up of three domain Cs H1, CH2 and CH3.

" light chain " is made up of light chain variable district (VL) and constant region of light chain (CL).

" constant region of light chain " is made up of a domain.

" weight chain variable district " and " light chain variable district " can be further subdivided into hypervariable region, be referred to as " complementary determining region " (CDR), CDR is dispersed in the more conservative region for being referred to as " framework region " (FR).Each VH and VL is by three CDR and four FR groups Into they are arranged in the following order from aminoterminal to c-terminus：FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, heavy chain and light The variable region of chain is contained can be with the binding structural domain of antigen interactions.

" constant region " can be included with the combination of mediated immunity globulin and host tissue or the factor, the host tissue or the factor The various cells (such as effector cell) of immune system and the first composition (C1q) of classical complement system.

" antigen-binding portion thereof " refers to retain one or more with the antibody of the ability of antigen (such as CD19) specific binding Individual fragment.Being proved the antigen binding function of antibody can be exercised by the fragment of full length antibody.Wrapped in " antigen-binding portion thereof " The binding fragment included includes：(1) Fab fragments, i.e., the monovalent fragment being made up of VL, VH, CL and CH1 domain；(2)F(ab’)₂ Fragment, i.e., two Fab fragments for passing through disulfide bond included in hinge area (region between heavy chain CHl and CH2 functional areas) Divalent fragments thereof；(3) the Fd fragments being made up of VH and CH1 domains；(4) Fv being made up of VL the and VH domains of antibody single armed Fragment；(5) the dAb fragments being made up of VH domains；(6) complementary determining region (CDR) of separation.In addition, although the two of Fv fragments Individual domain VL and VH is by single gene code, but they can be linked together using recombination method, can be made one The pairing of protein chain, wherein VL and VH areas forms monovalent molecule and is referred to as scFv (i.e. scFv), and this single-chain antibody is also included within In term " antigen-binding portion thereof ".These antibody fragments to be obtained with well known to a person skilled in the art routine techniques, and with it is complete Whole antibody identical method is screened to the practicality of these fragments.

" monoclonal antibody " refers to the antibody molecule preparation by single molecular composition.Monoclonal antibody combination is shown pair The specific binding of defined epitope (antigen part identified by antigen receptor specificity) and compatibility.

" Chimeric antigen receptor " (origin comes from the light chain (VL) and heavy chain (VH) of monoclonal antibody by extracellular antigen binding domain Composition, centre connected by the hinge area with toughness formed single-chain antibody (single chain fragment variable, ScFv), trans-membrane region and intracellular signal transduction district's groups into.

" Chimeric antigen receptor T cell ", i.e. CAR-T cells, turned by the single-stranded variable region and T cell signal of monoclonal antibody Lead area to be formed by connecting, antibody can be made after combining with corresponding tumour antigen with major histocompatibility complex non-limiting way T cell activation, and then play GVT.

" cell surface receptor " includes being capable of reception signal and the molecule and molecule across this signal of cytoplasma membrane propagation Compound.CD19 acceptors of the present invention are a kind of cell surface receptors.

" ka " refers to the combination of specific antibodies-antigen interactions, and " kd " refers to the solution of specific antibodies-antigen interactions From " KD " refers to dissociation constant, and it is to obtain (i.e. kd/ka) by kd and ka ratio, is represented with molar concentration (M).Antibody The conventional method that KD values can be established with this area determines, measure antibody K_DA kind of method for optimizing be surface plasmon resonance Method, preferably using bio-sensor system.

Technical scheme is described further below in conjunction with accompanying drawing and experimental example.

Portion of material source is illustrated in this：

CD19 albumen：Human CD19 (are purchased from Acrobiosysterms, article No. CD9-H8259；

The phage antibody library of people's single-chain antibody：(taking Loews medicine company Science and Technology Ltd. structure for Changzhou).

Streptavidin MagneSphere：(being purchased from Invitrogen companies).

Enzyme linked immunological microwell plate：The low flat bottom microtiter plate of 96 half bore (is purchased from Corning companies).

Anti-M13HRP antibody：(being purchased from Thermo Fisher companies).

M13 helper phages：(being purchased from Invitrogen companies).

SOC culture mediums：(purchased from Shanghai life work)

PCGMT phage vectors：(being purchased from Addgene companies)

XL1-blue bacteriums：(being purchased from Agilent company, article No. 200228).

LB solid medium flat boards：5g yeast extracts, 10g peptones, 10g sodium chloride, 10g agar powders are dissolved in 1L's In distilled water, the autoclaving under 121 DEG C of high temperature, then it is poured on flat board.

Developer solution ABTS solution：(being purchased from Thermo Fisher companies, article No. 002024).

Gelred nucleic acid dyes：(being purchased from Thermo Fisher companies).

PFUSE expression vectors：(being purchased from Invitrogen companies).

Plasmid extraction kit：(being purchased from QIAGEN companies).

Restriction enzyme：(being purchased from Takara companies).

Recombinase：(being purchased from Novoprotein companies).

293Fectin transfection reagents：(being purchased from Invitrogen companies, article No. 12347500).

293Freestyle suspension cells：(being purchased from Thermo Fisher companies).

Immunoglobulin IgG1 constant region fc section：(being purchased from Nanjing Jin Sirui companies).

BCA method protein quantification kits：(being purchased from Pierce, article No. 23252).

CBS antigen fixed solutions：By 1.59g Na₂CO₃With 2.93g NaHCO₃It is dissolved in 1L water, adjustment pH value is 9.6.

Anti-Human Fc HRP secondary antibodies：(being purchased from Thermo Fisher companies).

The saturating plate of 96 bottom holes：(being purchased from Corning companies).

Biacore instruments：(being purchased from GE companies, T200).

Protein A chips：(being purchased from GE companies).

The screening of the anti-CD19 single-chain antibodies of experimental example 1

1st, the phage antibody library of full human single chain variable fragments antibody is established

The weight chain variable district and light chain variable district of primer amplification antibody are designed, is used by way of Overlap extension PCR Linker connects weight chain variable district and light chain variable district, obtains the PCR primer of total length, with SfiI digestions PCR primer and PCGMT phagemid vectors, connection converted product electricity is transformed into XL1-blue competent cells, then added into competent cell Enter 3mL SOC culture mediums, after 30 DEG C are cultivated 1h, add final concentration of 50 μ g/mL ampicillin, 10 μ g/mL tetracycline Solution 20mL, 37 DEG C of shaken cultivation 2h.Then it is 10 to add concentration¹³/ mL μ the L of VCSM13 helper phages 50, incubation at room temperature 1h, centre every 10min jogs once.37 DEG C of shaken cultivation 2h, the card for adding final concentration of 70 μ g/mL receive mycin, 30 DEG C It is incubated overnight.Supernatant is collected by centrifugation, adds the PEG-8000/ sodium chloride solutions (PEG is polyethylene glycol) that concentration is 10%, is placed in 1h, 8000rpm, 4 DEG C of centrifugations of ice bath, abandon supernatant, with the PBS solution (phosphorus for the BSA (bovine serum albumin(BSA)) that 2mL concentration is 1% Acid buffer) precipitation is fully dissolved, supernatant, the phage antibody library of as full people's single-chain antibody is collected by centrifugation.

2nd, antibody screening

200 μ L are taken (containing bacteriophage 1 × 10¹²/ mL) the full human single chain variable fragments antibody of expression phage antibody library and 5 μ g CD19 antigens mix, and incubation at room temperature adds 50 μ L Streptavidin MagneSphere after 2 hours, the bacteriophage with antigen binding is by strepto- Avidin magnetic bead is captured, and uncombined bacteriophage is gone after 0.5%Tween-20 PBS solution (phosphate buffer) rinsing Remove, will be stand-by with the bacteriophage elution of magnetic bead stable bond with glycine hydrochloride solution (pH2.2).

XL1-Blue bacterium 20mL are inoculated with, treat OD₆₀₀After (light absorption value at 600nm wavelength, optical density) reaches 0.6, add The bacteriophage of above-mentioned elution, 30min is stood at 37 DEG C with XL1-Blue bacteriums, bacterium solution is coated on amicillin resistance flat board On, the thalline in ammonia benzyl resistant panel is collected in elution in second day, is infecting 1 × 10¹²Pfu/mL (pfu, plaque forming Unit, plaque-forming unit) M13 helper phages after, after amplification carry out next round screening, altogether carry out 3 wheel screening.

The XL1-Blue bacterium solutions for infecting bacteriophage are fully diluted, this bacterium solution then is coated on into a diameter of 15cm resistances is The LB solid medium flat boards of ampicillin, there are 100-500 clone, picking monoclonal antibody, by biting on each flat board Thalline enzyme linked immunoassay is verified to each wheel phage library after elutriation.

3rd, bacteriophage enzyme linked immunoassay

The 96 hole Bacteria Culture plates that the XL1-Blue monoclonal bacterium for transfecting bacteriophage are inoculated into 2mL are (public purchased from Corning Department) in, the SB culture mediums that 500 μ L contain tetracyclin resistance are added, the lower 37 DEG C of shaking table 4-6h of 200rpm rotating speeds, detect OD₆₀₀Value connects After nearly 0.6,1 μ L helper phage is added, the incubator overnight at 30 DEG C, 3000g centrifuging and taking supernatants are stand-by within second day.

Enzyme linked immunological microwell plate is taken, 4 DEG C of envelope antigen is stayed overnight, the 3rd day, PBST (phosphate buffer containing Tween-20) The rear enclosed of elution, the bacteriophage supernatant that previous step prepares then is added, be incubated at room temperature 2h, PBST adds Anti- M13HRP antibody incubation 30min, after washed 3 times with PBST, add 50 μ L developer solutions ABTS.

4th, experimental result：

As shown in figure 1, to screen the bar chart of the single-stranded scFv antibody fragments results of CD19 in experimental example 1, table 1 is experimental example The single-stranded scFv antibody fragments results of CD19 are screened in 1.

Table 1 is that the single-stranded scFv antibody fragments results of CD19 are screened in experimental example 1

。

From Fig. 1 and table 1 as can be seen that CD19 antigens sieve in the phage antibody library of full human single chain variable fragments antibody by 3 wheels After choosing, third round CD19 antigen selections result shows that monoclonal number is 82 times of the first round, and showing being capable of table after 3 wheel screenings Constantly it is enriched with up to the bacteriophage with CD19 antigentic specificity binding antibodies.In the phage library obtained after the 3rd wheel screening 120 monoclonals of picking carry out enzyme linked immunoassay checking, finally determine that the signal intensity of enzyme linked immunoassay is more than 1 Dan Ke It is grand to be used as positive monoclonal, positive monoclonal is sequenced, nucleotide coding sequence is obtained, the sequence of acquisition is compared Analysis, repeated cloning is more, illustrates that the affinity of the antibody sequence may be stronger, the nucleotide coding being effectively enriched with this determination Sequence.

The preparation (expression and purifying) of the anti-CD19 single-chain antibodies of experimental example 2

The monoclonal plasmid that will screen to obtain in experimental example 1, pass through weight after being connected after the digestions of restriction enzyme Sfi I By fragment access pFUSE expression vectors, (pFUSE expression vectors act on digestion position to the method for group by restriction enzyme Sfi I Point) in, be derived from the present invention antibody pFUSE expression vectors.

According to volume mass ratio it is 30 μ L by the eucaryon antibody expression vector of 293Fectin transfection reagents and above-mentioned acquisition： 30 μ g ratio mixing, add 30 μ L 293Freestyle suspension cells, 37 DEG C of shaking table culture 48-72 under 125rpm rotating speeds Hour, collected after centrifugation supernatant, existed using HiTrap Protein A HP columnsAlbumen is pure Change and antibody protein purifying is carried out on instrument, anti-CD19 single-chain antibodies (the anti-full people source scFv antibody of CD19) are obtained, referring finally to BCA methods The specification detection antibody concentration of protein quantification kit.

Experimental example 3 detects the binding specificity and binding kineticses feature of the full people source scFv antibody of anti-CD19

Fix to use and catch with antigen affinity and dynamic characteristic, antibody using the dynamic (dynamical) method detection antibody of multi-cycle Method progress is obtained, first anti-CD19 antigens are coupled on sensor (sensor), diluted concentration is then complete by anti-CD19 to 5 μ g/mL Chip surface is flowed through after the scFv samples dilution of people source, it is 7.5 μ g/mL and 15 μ g/mL, the anti-full people source scFv of CD19 to be diluted to concentration Antibody will be captured by CD19 antigens, and antigen is detected and recorded with signal after antibody binding, finally uses regenerative agent (pH1.5 glycine solution) all elutes the antibody of sensor surface and antigen samples, and carries out new round detection.

Experimental result：

As shown in Fig. 2 being the full people source scFv antibody of anti-CD19 and CD19 binding kineticses curve maps, each line represents not The same full people source scFv antibody concentrations of anti-CD19, respectively 7.5 μ g/mL and 15 μ g/mL.Anti-CD 19 antibodies concentration is higher, concentration What is respectively combined is faster, and signal is stronger, illustrates that the full people source scFv antibody of anti-CD19 and CD19 affinity are good, rearmounted at 120 seconds Change in PBS solution, by antigen and antibody dissociation, the full human monoclonal antibodies of anti-CD19 and CD19 are proved by Octet detections Protein-interacting, the full people source scFv of anti-CD19 of the invention K_DFor 2 × 10^-6M。

Experimental example 4 targets CD19 CAR-T cells and application

1st, prepared by PBMC separation：

Healthy human peripheral blood is gathered with anticoagulant tube, dilutes anticoagulation (1 with PBS:1) it is slowly added to after equipped with isometric Lymphocyte separation medium (Ficoll) 50mL centrifuge tubes in, it is slow to rise slow drop centrifugation 25min with 450 (× g) centrifugal force.

After centrifugation terminates, the careful tunica albuginea layer drawn above lymphocyte separation medium, a new 50mL centrifuge tube is transferred to In, PBS is added, it is slow to rise slow drop centrifugation 10min with 300 (× g) centrifugal force, supernatant is abandoned, the cell for retaining centrifugation bottom of the tube sinks Form sediment；PBS is added again, it is slow to rise slow drop centrifugation 15min with 160 (× g) centrifugal force, abandon supernatant；PBS is eventually adding, with 300 (× g) centrifugal force, it is slow to rise slow drop centrifugation 10min, abandon supernatant, that is, obtain PBMC.

2nd, CAR T cells are prepared

The PBMC of above-mentioned preparation is slightly cultivated 1 to 2 with T cell culture medium (culture mediums of RPMI 1640 containing 10%FBS) Carry out electricity after individual hour to turn, transfection is following two groups respectively：

(1) as shown in figure 3, being expressed for the CD19-C8scFv-CAR piggyBac transposon used in the present invention (T2A adjusts peptide, puromycin-resistant Puro to the structure chart of carrier^R, costimulatory signal area is functional signal conducting structure domain), By each 5 μ of CD19-C8scFv-CAR two plasmids of piggyBac transposon and Super piggyBac transposase G turns buffer solution mixing with the electricity in human T-cell's consideration convey transfection reagent box (Lonza, VPA-1002), obtains comprising two kinds of plasmids About 110 μ L electricity turn mixed liquor；

(2) to include original plasmid CD19-CAR piggyBac transposon and Super piggyBac The electricity of two plasmids of transposase turns mixed liquor as control；A part of cell is separately stayed to turn without electricity.

Above-mentioned plasmid Super piggyBac transposase are from System Bioscience (Cat#:PB200PA- 1) the business plasmid of purchase.Plasmid CD19-C8scFv-CAR piggyBac transposon preparation method is referring to document Zhu,X.,et al.,Patient-derived glioblastoma stem cells are killed by CD133- specific CAR T cells but induce the T cell aging marker CD57.Oncotarget, 2015.6(1):p.171-184。

With reference to the specification of human T-cell's consideration convey transfection reagent box (Lonza, VPA-1002), turn mixed with described two groups electricity respectively Close liquid and hang 1-2 × 10⁷Individual PBMC cells carry out electricity and turned, and electricity turns that cell is transferred to the T cell culture medium of preheating at once after terminating； A subculture is changed again after 2 hours.

The T cell training of the IL-2 (Interleukin 2, interleukin 2) containing 100IU/mL is added after 16-18 hours Base is supported, the magnetic bead for having CD3/CD28 antibody with coupling (is purchased from ThermoFisher SCIENTIFIC, article No.：11141D) stimulate Activate CD3 positive T cells.

The IL-2 containing 100IU/mL more renewed for every 2 to 3 days T cell culture medium, while observe cell amplification situation.Root Determine that it stimulates duration according to its proliferative conditions, but stimulation time is no more than one week.

Separate except after magnetic bead, 1 μ g/mL puromycins of addition continue culture 5 to 7 days.A small amount of cell is collected to be used to detect, Remaining cell can be continuing with rapid amplification and further largely expand, and the CAR T for obtaining the targeting CD19 with C8scFv are thin Born of the same parents.

3rd, Flow cytometry CAR expression is utilized

By 10⁵Expand obtained CAR-T cells and be resuspended in 100 microlitres of FACS buffer solutions (EDTA containing 2mM and 0.5%BSA PBS) in (EDTA, i.e. Ethylenediaminetetraacetic acid), Myc-Tag is added in cell suspension (9B11)Mouse mAb(1:500 dilutions, Cell Signaling, 2276S), 4 DEG C are incubated 15 minutes；After cleaning cell once 100 microlitres of FACS buffer solutions are resuspended in, add the sheep anti mouse secondary antibody (1 of PE marks (phycoerythrin, phycoerythrin):50 is dilute Release, be purchased from：Jackson, article No.：115-116-072), 4 DEG C are incubated 10 minutes；CytoFLEX (is purchased from：Beckman Coulter) flow cytometer is used to obtain staining cell, and CytExpert is used for analysis result.

As shown in figure 4, the stream for CD19-C8scFv-CAR T cells surface C AR expression rates made from experimental example 4 of the present invention Formula testing result figure (NT, N-terminal cleavage product), flow cytometry show CD19-C8scFv-CAR- The high expression CD19-C8scFv-CAR molecules of T cell.

4th, CD19-C8scFv-CAR-T cell cytokines releasing result is assessed

The present invention is drenched with not expressing the chronic marrow original K562 Leukaemia of CD19 people and expressing CD19 people Burkitt ' s Bar oncocyte Raji trains above-mentioned two target cell as target cell altogether with effector cell CD19-C8scFv-CAR T cells respectively Support.

Respectively by target cell Raji or K562 with every hole 1x10⁵Kind is in 96 orifice plates, by effector cell CD19-C8scFv- CAR T cells are with 2 × 10⁵Mixed with target cell, with the μ L of cumulative volume 200 nutrient solution (culture medium+10%FBS of RPMI 1640) In 37 DEG C of 5%CO₂24 hours, every group of 3 multiple holes are co-cultured in incubator.The μ L of centrifuging and taking supernatant 10 are used afterwards PerkinElmer AlphaLISAassay reagents series box (AL208, AL216, AL217, AL221) detect respectively TNF-α, GM-CSF, IFN-γ and IL-2 cytokine concentrations are horizontal.

As shown in figure 5, trained altogether with target cell for obtained CD19-C8scFv-CAR-T cells point in the embodiment of the present invention 4 The secretory volume column diagram of cell factor IFN-γ after supporting, as shown in fig. 6, being obtained CD19- in the embodiment of the present invention 4 The secretory volume column diagram of C8scFv-CAR-T cells point and the cell factor IL-2 after target cell co-cultivation, as shown in fig. 7, being this Point of cytokine TNF-α after obtained CD19-C8scFv-CAR-T cells point co-culture with target cell in inventive embodiments 4 The amount of secreting column diagram, as shown in figure 8, being total to for obtained CD19-C8scFv-CAR-T cells point in the embodiment of the present invention 4 with target cell The secretory volume column diagram of cell factor GM-CSF after culture.From Fig. 5-8 as can be seen that cell factor IFN-γ (Interferon γ), IL-2 (Interleukin-2), TNF-α (tumor necrosis factor α) and GM-CSF The horizontal Raji targets in high expression CD19 of (granulocyte-macrophage colony stimulating factor) Cytositimulation group is significantly higher than the K562 target cell stimulation groups for not expressing CD19.Illustrate that CD19-C8scFv-CAR-T cells can be with Effectively activated.

In summary, anti-CD19 of the invention human antibody or antibody fragment and its methods and applications, it can be high Specific and people source CD19 protein binding, expression is engineered in T cell using Chimeric antigen receptor T cell technology and is integrated, Obtained Chimeric antigen receptor T cell can be used in treating the hematologic cancer related to CD19 expression.

Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.

<110>Take Loews medicine company Science and Technology Ltd. in Changzhou

<120>A kind of anti-CD19 human antibody or antibody fragment and its methods and applications

<160> 11

<170> PatentIn version 3.5

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

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Arg Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser

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Trp Ile Ala Asn Ile Arg Glu Arg Arg Gly Asn Thr Arg Tyr Ala Glu

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Ser Val Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Met Asn Ser

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Val Tyr Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Gly Ile Tyr

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Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

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Ser Gly Ser Lys Ser Arg Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

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Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

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atggcacagg tgcagctggt ggagactggg ggaggcttgg tcaagcctcg agggtccctg 60

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aaggctccag ggagggggct ggagtggatt gcaaacattc gcgagaggag aggcaataca 180

aggtacgcag agtctgtcaa gggccgattc accatcgcca gagacaacgc catgaactca 240

gtgtatctgc aaatgaacag cctgagagtc gaggacacgg gcatctatta ttgtgcgaga 300

agtcatagaa gtggctggtt cttggacgtc tggggccaag ggaccacggt caccgtctcc 360

tca 363

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cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

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cacccaggca aagcccccaa actcatgatt tatgatgtca gtaagcggcc ctcaggggtt180

tctaatcgct tctctggctc caagtctcgc aacacggcct ccctgaccat ctctgggctc240

caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactccctta 300

ttcggcggag ggaccaagct gaccgtccta ggt 333

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Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

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Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

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Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

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Ala Thr Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro

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Claims

1. a kind of anti-CD19 human antibody or antibody fragment, it is characterised in that the antibody or antibody fragment include：Heavy chain and Light chain；

Described heavy chain and light chain include variable region, and described variable region includes complementary determining region CDR1, CDR2 and CDR3；

Complementary determining region CDR1, CDR2 and CDR3 of described heavy chain are represented with HCDR1, HCDR2 and HCDR3 respectively；

Complementary determining region CDR1, CDR2 and CDR3 of described light chain are represented with LCDR1, LCDR2 and LCDR3 respectively；

Described HCDR1 amino acid sequence includes SEQ ID NO：3 or the sequence in one or a few amino acid mutation SEQ ID NO：3；

Described HCDR2 amino acid sequence includes SEQ ID NO：4 or the sequence in one or a few amino acid mutation SEQ ID NO：4；

Described HCDR3 amino acid sequence includes SEQ ID NO：5 or the sequence in one or a few amino acid mutation SEQ ID NO：5；

Described LCDR1 amino acid sequence includes SEQ ID NO：6 or the sequence in one or a few amino acid mutation SEQ ID NO：6；

Described LCDR2 amino acid sequence includes DVS；

Described LCDR3 amino acid sequence includes SEQ ID NO：7 or the sequence in one or a few amino acid mutation SEQ ID NO：7.

2. anti-CD19 according to claim 1 human antibody or antibody fragment, it is characterised in that described heavy chain can Becoming region amino acid sequence includes SEQ ID NO：1 or the sequence in one or a few amino acid mutation SEQ ID NO：1；

Described chain variable region amino acid sequence includes SEQ ID NO：2 or the sequence in one or a few amino acid dash forward The SEQ ID NO of change：2.

3. anti-CD19 according to claim 1 or 2 human antibody or antibody fragment, it is characterised in that described weight Chain represents that described light chain is represented with VL with VH；Described antibody or the amino acid sequence of antibody fragment also include VH- GGGGGS-VL and/or VL-GGGGGS-VH.

4. anti-CD19 according to claim 3 human antibody or antibody fragment, it is characterised in that described antibody or Antibody fragment is with 2 × 10^-6M or lower KD values and people's CD19 protein bindings.

5. a kind of nucleotides, it is characterised in that anti-CD19 of the nucleotide coding as described in any one in claim 1-4 Human antibody or antibody fragment；

Described nucleotides includes：For encoding the nucleotide sequence SEQ ID NO of described weight chain variable district：8, and be used for The nucleotide sequence SEQ ID NO of the described light chain variable district of coding：9.

6. a kind of Chimeric antigen receptor, it is characterised in that this receptor contains anti-as described in any one in claim 1-4 CD19 human antibody or antibody fragment.

7. Chimeric antigen receptor according to claim 6, it is characterised in that this receptor includes what is be sequentially connected：Signal peptide Sequence, CD19 binding structural domains, detection label, hinge area and membrane spaning domain, and functional signal conducting structure domain；

Described CD19 binding structural domains include scFv, and the scFv includes anti-CD19 human antibody or antibody piece as mentioned Section；

Described hinge area and membrane spaning domain includes：Hinge area Hinge TM and transmembrane structure CD8, the hinge area Hinge TM Include SEQ ID NO with transmembrane structure CD8 amino acid sequence：10 or the sequence in one or a few amino acid mutation SEQ ID NO：10；

Described CD28 amino acid sequence includes SEQ ID NO：11 or the sequence in one or a few amino acid mutation SEQ ID NO：11；

Described 4-1BB amino acid sequence includes SEQ ID NO：12 or the sequence in one or a few amino acid mutation SEQ ID NO：12；

Described CD3Zeta amino acid sequence includes SEQ ID NO：13 or the sequence in one or a few amino acid dash forward The SEQ ID NO of change：13；

Described signal peptide sequence includes CSF2RA, and its amino acid sequence includes SEQ ID NO：14 or the sequence in one or few The SEQ ID NO of the several amino acid mutations of number：14；

Described detection label includes C-Myc, and its amino acid sequence includes SEQ ID NO：15 or the sequence in one or a small number of The SEQ ID NO of several amino acid mutations：15.

8. a kind of T cell for being used to treat or prevent tumour, it is characterised in that the T cell contains as claimed in claims 6 or 7 Chimeric antigen receptor.

A kind of 9. method for preparing Chimeric antigen receptor, it is characterised in that this method contains such as claim 6 using transposons structure Or the T cell of the Chimeric antigen receptor described in 7.

A kind of 10. method for detecting effector cell and being activated, it is characterised in that this method includes：The tumour for expressing CD19 is thin Born of the same parents and do not express CD19 tumour cell and be used as target cell, be expressed as expressing CD19 groups and do not express CD19 groups, by table Up to Chimeric antigen receptor CD19-C8scFv-CAR-T cells as effector cell, CD19 components are not expressed expression CD19 groups and Do not co-cultured with effector cell, detect cell factor IFN-γ, IL-2, TNF-α and GM-CSF level；

If the level for the cell factor that CD19-C8scFv-CAR-T cells are secreted, which is higher than, in CD19 groups are expressed does not express CD19 Group, described CAR-T effector cell are activated；

Described CD19-C8scFv-CAR-T cells are the T cell as claimed in claim 8 for being used to treat or prevent tumour.