CN106589129A - Three-function molecule combining CD19, CD3 and CD28 and application of three-function molecule - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a three-function molecule combining CD19, CD3 and CD28 and application of the three-function molecule. The three-function molecule capable of identifying CD19, CD3 and CD28 simultaneously is constructed through a genetic engineering method and an antibody engineering method. In the aspects of a preparation technology and practical application, the molecule has the obvious advantages that the effect of activated T cells is further improved while the targeting property on CD19 positive cells is given to the T cells; when only CD19 is added, the bonding and killing effects of the CD19-mediated T cells on CD19 positive target cells are superior to those of an anti-CD19/anti-CD3 BiTE bispecific antibody, and the using convenience is superior to that of a CD19-targetted CAR-T technology.

Description

Three functional moleculars of a kind of combination CD19, CD3 and CD28 and its application

Technical field

The invention belongs to biomedicine technical field, and in particular to a kind of three functional moleculars of combination CD19, CD3 and CD28 And its application.

Background technology

Mankind's CD19 antigens are the transmembrane glycoproteins that size is 95kDa, are subordinated to immunoglobulin superfamily, except being expressed in Normal B lymphocytes surface, CD19 is also high to be expressed in B cell malignant tumor, therefore anti-CD19 monoclonals full length antibody is opened Send out and be applied to treat urgency/chronic lymphocytic leukemia and B cell lymphoma (Wang K et al., Experimental Hematology＆Oncology, 1:36-42,2012).In view of anti-CD19 monoclonal antibodies cannot effectively raise cytotoxic T pouring (Cytotoxic T lymphocyte, CTL, the double positive T cells of such CD3/CD8 can specific recognition target cell table for bar cell The antigenic peptides in face/MHC I MHC molecule complex, discharge perforin (Peforin), cause target cell lysis dead after autoactivation Die, also can secretory cell toxin and granzyme (Granzyme) etc. cause the DNA damage of target cell core, cause target cell apoptosis), People further design and develop the bi-specific antibody (Bi-specific that can be connected T cell and lymphoma B cell Antibody, BsAb) and genetic engineering Chimeric antigen receptor T cell immunotherapy (Chimeric antigen receptor T-cell immunotherapy, CAR-T) (Zhukovsky EA et al., Current Opinion in Immunology, 40:24-35,2016).

A kind of bi-specific antibody type of the targeting CD19 of comparative maturity is the bispecific T of anti-CD19/ AntiCD3 McAbs at present Cell adapter (Bi-specific T cell engager, BiTE), its structure is two single-chain antibody (Single-chain Variable fragment, scFv) by having, flexible connection fragments of peptides (Linker) is covalent to connect functional domain (Goebeler ME et al., Leukemia＆Lymphoma, 57：1021-1032,2016).In the cellular immune processes of body, The TCR/CD3 complex on CD8 positive T cells surface and antigen presenting cell (Antigen presenting cell, APC) table There is specific recognition in the endogenous antigen peptide in face/MHC I MHC molecule complex, cause the kytoplasm section phase of CD3 and co-receptor CD8 Interaction, activates the protein tyrosine kinase being connected with kytoplasm segment trailer, swashs CD3 cytoplasmic regions immunity receptor tyrosine kinase Tyrosine phosphorylation in die body (Immunoreceptor tyrosine-based activation motif, ITAM) living, Enabling signal transduction molecule cascade reaction, activating transcription factor so that T cell primary activation.Anti- CD19/ AntiCD3 McAbs BiTE is double special Heterogenetic antibody can be formed between T cell and neoplastic B cell due to the binding activity with the mankind's two kinds of antigens of CD3 and CD19 Cell is connected, while giving T cell primary activation signal, improves its killing targeting to tumor cell.But, BiTE is double Specific antibody Fc fragments with full length antibody, molecular weight of albumen less (～54kDa), therefore in the process of oncotherapy In can pass through hematuria barrier and brain blood barrier, bioavailability is low, needs persistent intravenous injection to be administered, while with certain nerve Toxicity.

Additionally, the activation of T cell needs to rely on dual signal pipeline (Baxter AG et al., Nature in human body Reviews Immunology, 2：439-446,2002).First, the antigenic peptides of APC cell surfaces-MHC molecule complex and T The TCR/CD3 complex of cell surface interacts and produces the first signal, afterwards the costimulatory moleculeses on antigen presenting cell surface Part (such as CD80, CD86) is interacted with the costimulatory moleculeses (such as CD28) on T cell surface and produces secondary signal.It is existing Research show only the first signaling pathways cannot abundant activating T cell, the even generation activation of its disability can be caused on the contrary to lure T cell death (Activation induced cell death, AICD) led.To solve this problem, can be by anti-tumor The bi-specific antibody of original/anti-CD28 is used in combination with the bi-specific antibody of tumor-resistant antigen/AntiCD3 McAb, to improve T cell Activation and tumor cytotoxicity efficiency (Jung G et al., Int J Cancer, 91：225-230,2001；Kodama H et al., Immunol Lett, 81：99-106,2002).But the method has inconvenience in actual mechanical process, for example, can increase The workload and production cost of restructuring bi-specific antibody expression and purification, also needs two kinds of optimization double special during activation amplification T cell The relative scale of heterogenetic antibody.By contrast, CAR-T technologies can preferably solve the problems, such as the activation of T cell.The structure of CAR leads to Often include：(such as CD19 antigen binding domains are typically derived from anti-CD19 monoclonals full length antibody for tumor associated antigen land ScFv fragments), extracellular hinge region, transmembrane region and intracellular signal area.Wherein intracellular signal area is responsible for the activation of mediate T cell, One side completes the first stimulus signal by the Tyrosine Activating Motifs on CD3 ζ chains, on the one hand by CD28 costimulatory signals The expansion of the first stimulus signal is realized, is promoted T cell propagation and activation, and is caused cytokine secretion increase, anti-apoptotic Protein excretion increases, cell death postpones etc..But CAR-T technologies there is also some shortcomings in itself：First, the technology relies on disease Poison transfection carries out genetic modification to T cell, and complex steps are required to experiment condition higher；Secondly, need in vitro when specifically used CAR-T cells after amplifying activated are fed back in patient body, and dosage control Relative antibody medicine has bigger difficulty；Additionally, CAR-T cells rear quantity in patient's body is sharply increased and can cause cytokine storm (Cytokine storm), short-term The interior cytokine for producing excess, so as to cause the even side reaction such as death of hyperpyrexia, low pressure, shock.

The content of the invention

In order to overcome the problem in the presence of prior art, it is an object of the invention to provide one kind in combination with CD19, Three functional moleculars of CD3 and CD28 and its application.

To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：

A first aspect of the present invention, there is provided a kind of three functional molecular, its structure include being incorporated into the first of CD19 Functional domain, can with reference to and activate T cell surface C D3 molecule the second functional domain and can with reference to and activate T cell surface 3rd functional domain of CD28 molecules.

Preferably, three functional molecular can recognize CD19 while, with reference to and activate T cell surface C D3 molecule With CD28 molecules, so as to produce the first signal and secondary signal needed for T cell activation.

Preferably, first functional domain is the antibody of anti-CD19, and second functional domain is the antibody of AntiCD3 McAb, described 3rd functional domain is the antibody of anti-CD28.

Preferably, the antibody is small molecular antibody.

Preferably, the antibody is selected from Fab antibody, Fv antibody or single-chain antibody (scFv).

Preferably, connected by junction fragment 1 between first functional domain and second functional domain, second work( Can be connected by junction fragment 2 between domain and the 3rd functional domain.

Preferably, the junction fragment 1 and junction fragment 2 are selected from the junction fragment or immunoglobulin in units of G4S The hinge region fragment of IgD.

The G4S is specially GGGGS.The junction fragment in units of G4S includes one or more G4S units.Example Such as, the G4S units for being, two, three or more than four can be included.In some embodiments of the present invention, one is listed In the bifunctional molecule of monomeric form, connected by the junction fragment 1 in units of G4S between the first functional domain and the second functional domain Connect, connected by the junction fragment 2 in units of G4S between the second functional domain and the 3rd functional domain.The junction fragment 1 contains One G4S unit, the aminoacid sequence of the junction fragment is as shown in SEQ ID NO.23.The junction fragment 2 contains three G4S units, the aminoacid sequence of the junction fragment is as shown in SEQ ID NO.25.

The hinge region fragment of the Immunoglobulin IgD can be the hinge Ala90-Val170 of Immunoglobulin IgD.This In some embodiments of invention, list in the bifunctional molecule of a dimeric forms, the first functional domain and the second functional domain it Between connected by the junction fragment 1 in units of G4S, between the second functional domain and the 3rd functional domain pass through Immunoglobulin IgD The connection of hinge region fragment, the hinge Ala90- of the hinge region fragment of the Immunoglobulin IgD for Immunoglobulin IgD Val170.The junction fragment 1 contains a G4S unit, the aminoacid sequence such as SEQ ID NO.27 institutes of the junction fragment Show.The aminoacid sequence of the junction fragment 2 is as shown in SEQ ID NO.29.The junction fragment 2 can be mutual by disulfide bond Connection forms dimer.

Preferably, the C-terminal of first functional domain is connected with the N-terminal of second functional domain；Second function The C-terminal in domain is connected with the N-terminal of the 3rd functional domain.

Preferably, first functional domain is the single-chain antibody of anti-CD19, and second functional domain is the single-stranded anti-of AntiCD3 McAb Body, the 3rd functional domain is the single-chain antibody of anti-CD28, and the single-chain antibody includes weight chain variable district and light chain variable district.

Preferably, the aminoacid sequence of the weight chain variable district of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.6. The aminoacid sequence of the light chain variable district of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.7.The AntiCD3 McAb it is single-stranded The aminoacid sequence of the weight chain variable district of antibody is as shown in SEQ ID NO.9.The light chain variable district of the single-chain antibody of the AntiCD3 McAb Aminoacid sequence as shown in SEQ ID NO.10.The aminoacid sequence of the weight chain variable district of the single-chain antibody of the anti-CD28 is such as Shown in SEQ ID NO.12.The aminoacid sequence of the light chain variable district of the single-chain antibody of the anti-CD28 such as SEQ ID NO.13 institutes Show.

In some embodiments of the invention, the aminoacid sequence such as SEQ ID of the single-chain antibody of the anti-CD19 are listed Shown in NO.5.The aminoacid sequence of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.8.The anti-CD28's is single-stranded anti- The aminoacid sequence of body is as shown in SEQ ID NO.11.

In some embodiments of the invention, the aminoacid sequence such as SEQ ID of three functional moleculars of monomeric form have been also listed Shown in NO.1.The aminoacid sequence of three functional moleculars of dimeric forms is as shown in SEQ ID NO.3.

A kind of a second aspect of the present invention, there is provided polynucleotide, its aforementioned three functional molecular of coding.

A kind of a third aspect of the present invention, there is provided expression vector, which contains foregoing polynucleotides.

A kind of a fourth aspect of the present invention, there is provided host cell, which is converted by foregoing expression vectors.

A kind of a fifth aspect of the present invention, there is provided method for preparing aforementioned three functional molecular, including：Build containing three functions Then expression vector containing three functional molecular gene orders is converted and is lured into host cell by the expression vector of molecular gene sequence Expression is led, is separated from expression product and is obtained three described functional moleculars.

In the preferable case of the present invention, the expression vector adopts pcDNA3.1.The host cell adopts Chinese hamster Gonad cell (Chinese hamster ovary ce1l, CHO).

A sixth aspect of the present invention, there is provided aforementioned three functional molecular is used for the purposes for preparing anti-tumor medicine.

A seventh aspect of the present invention, there is provided a kind of cancer therapeutics compositions, containing aforementioned three functional molecular and at least A kind of pharmaceutically acceptable carrier or excipient.It is the positive tumors of CD19 that the tumor is cell surface.

A eighth aspect of the present invention, discloses a kind of method of external treatment tumor, including by aforementioned three functional molecular or Cancer therapeutics compositions are applied to tumor patient.Methods described can be non-treatment purpose.The tumor is cell table Face is the positive tumors of CD19.

Compared with prior art, the present invention has the advantages that：

(1) three of the invention functional moleculars be possible to be incorporated into first functional domain of CD19, can with reference to and activate T cell Second functional domain of surface C D3 molecule and can with reference to and activate the 3rd functional domain of T cell surface C D28 molecule and be blended in together One protein peptide chain, is produced using eukaryotic cell expression system, and expression product structure is single, and purifying process is easy, and protein yield is high, Preparation technology and product are stable, easy to use；And anti-CD19/ AntiCD3 McAbs bi-specific antibody CD28 bispecifics anti-with anti-CD19/ If antibody is used in combination, two bi-specific antibodys need to distinguish expression and purification, and preparation technology is more complicated, workload and produce into Originally dramatically increase, and both relative scales during use, need to be optimized.

(2) three functional moleculars of the invention can produce the second stimulus signal of T cell activation, give T cell targeting Property while further increase the activation effect to T cell so that cytokine and Anti-apoptotic proteins secretion increase, have Effect avoids the phenomenon of T cell disability and death, and combination of the T cell for being mediated to CD19 positive target cells can be reached with killing To the effect of even better than anti-CD19/ AntiCD3 McAbs BiTE bi-specific antibodys, and albumen consumption is less.

(3) three functional molecular of the present invention is compared with the CAR-T technologies of targeting CD19, is not related to virus-mediated turn base Because of operating procedures such as, ex vivo T cell culture and feedbacks, using more convenient, dosage is controllable, cause into after patient's body cell because The risk of sub excessive release is little, it is to avoid using toxic and side effects during CAR-T.

Description of the drawings

Fig. 1：A. the knot of the anti-CD28 three-specific antibodies of the anti-CD19/CD3/ of monomeric form (CD19-CD3-CD28 TsAb_M) Composition；B. the structure of the anti-CD19/ AntiCD3 McAbs of dimeric forms/anti- CD28 three-specific antibodies (CD19-CD3-CD28TsAb_D) Figure.

Fig. 2：A. CD19-CD3-CD28 TsAb_M SDS-PAGE analysis charts of purification, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD19-CD3-CD28 TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-CD28TsAb_M；B. The CD19-CD3-CD28 TsAb_D SDS-PAGE analysis charts of purification；Swimming lane 1：Molecular weight protein Marker；Swimming lane 2：Reproducibility CD19-CD3-CD28 TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-CD28TsAb_D.

Fig. 3 A：The ELISA qualification results of CD19-CD3-CD28 TsAb_M, the curve in figure represent 4 kinds of detection knots respectively Really：■ is coated with 1 μ g/ml recombinant antigen CD19-hFc；● 1 μ g/ml recombinant antigen CD3-hFc of coating；The 1 μ g/ml restructuring of ▲ coating Antigen CD28-hFc；The measurement result of any antigen is not coated with.

Fig. 3 B：The ELISA qualification results of CD19-CD3-CD28 TsAb_D；Curve in figure represents 4 kinds of detection knots respectively Really：■ is coated with 1 μ g/ml recombinant antigen CD19-hFc；● 1 μ g/ml recombinant antigen CD3-hFc of coating；The 1 μ g/ml restructuring of ▲ coating Antigen CD28-hFc；The measurement result of any antigen is not coated with.

Fig. 4：Three specific antibodies and Mediated by Bi-specific Antibodies cell linking experiment, using Raji lymphoma cells as CD19 positive target cell, Jurkat cell detect variable concentrations CD19- respectively as CD3 and CD28 positive effector lymphocyte The cell linking activity of CD3-CD28 TsAb_M, CD19-CD3-CD28 TsAb_D and CD19-CD3 BsAb；A：It is not added with The blank of antibody；B：The experimental group of addition high concentration CD19-CD3 BsAb (45ng/ml)；C：Addition high concentration CD19- The experimental group of CD3-CD28TsAb_M (45ng/ml)；D：The reality of addition high concentration CD19-CD3-CD28TsAb_D (45ng/ml) Test group；E：The experimental group of concentration C D19-CD3BsAb (0.45ng/ml) in addition；F：Concentration C D19-CD3- in addition The experimental group of CD28TsAb_M (0.45ng/ml)；G：The reality of concentration C D19-CD3-CD28TsAb_D (0.45ng/ml) in addition Test group；H：The experimental group of addition low concentration CD19-CD3BsAb (0.0045ng/ml)；I：Addition low concentration CD19-CD3- The experimental group of CD28TsAb_M (0.0045ng/ml)；J：Addition low concentration CD19-CD3-CD28TsAb_D (0.0045ng/ml) Experimental group.

Fig. 5 A：Three specific antibodies and Mediated by Bi-specific Antibodies cell killing experiment, using Raji lymphoma cells as CD19 positive target cell, CIK (Cytokine induced killer) cells are used as CD3 and CD28 positive lethal effect Cell, detects variable concentrations CD19-CD3-CD28TsAb_M, CD19-CD3-CD28TsAb_D and CD19-CD3BsAb respectively Killing-efficiency of the CIK cell for being mediated to Raji cells；Effector lymphocyte：Target cell (E:T ratios)=1：5, kill the time：3h.

Fig. 5 B：Three specific antibodies and Mediated by Bi-specific Antibodies cell killing experiment, using Raji lymphoma cells as CD19 positive target cell, CIK cell detect variable concentrations CD19- respectively as CD3 and CD28 positive lethal effect cell The CIK cell mediated by CD3-CD28TsAb_M, CD19-CD3-CD28TsAb_D and CD19-CD3BsAb is to Raji cells Killing-efficiency；Effector lymphocyte：Target cell (E:T ratios)=1：1, kill the time：3h.

Specific embodiment

First, term and abbreviation：

CTL：Cytotoxic T lymphocyte (Cytotoxic T lymphocyte)

BsAb：Bi-specific antibody (Bi-specific Antibody)

TsAb：Three-specific antibody (Tri-specific Antibody)

BiTE：Bispecific T cell adapter (Bi-specific T cell engager)

TiTE：Tri-specific T cell adapter (Tri-specific T cell engager)

Fab：Fab (Fragement of antigen binding)

Fv：Variable region fragment (Variable fragment)

scFv：Single chain variable fragment (Single-chain variable fragment), is also called single-chain antibody

V_H：Weight chain variable district (Heavy chain variable region)

V_L：Light chain variable district (Light chain variable region)

Linker：Junction fragment

Linker1：Junction fragment 1

Linker2:Junction fragment 2

CD19-CD3-CD28TsAb_M：The anti-CD19/ AntiCD3 McAbs of monomeric form/anti- CD28 three-specific antibodies

CD19-CD3-CD28TsAb_D：The anti-CD19/ AntiCD3 McAbs of dimeric forms/anti- CD28 three-specific antibodies

2nd, three functional molecular

Three functional molecular of the present invention, its structure include the first functional domain, the Neng Goujie that can be incorporated into CD19 Merge activation T cell surface C D3 molecule the second functional domain and can with reference to and activate the 3rd work(of T cell surface C D28 molecule Can domain.

Further, three functional molecular can while CD19 is recognized, with reference to and activate T cell surface C D3 point Son and CD28 molecules, so as to produce the first signal and secondary signal needed for T cell activation.

The present invention has no particular restriction for the first functional domain, the second functional domain and the 3rd functional domain, as long as can know While other CD19, with reference to and activate T cell surface C D3 molecule and CD28 molecules, so as to produce first needed for T cell activation Signal and secondary signal.For example, first functional domain can be the antibody of anti-CD19, and second functional domain can be The antibody of AntiCD3 McAb, the 3rd functional domain can be the antibody of anti-CD28.The antibody can be arbitrary form.But either The antibody of which kind of form, its antigen-binding site contain weight chain variable district and light chain variable district.The antibody preferably can be with It is small molecular antibody.The small molecular antibody is the antibody fragment of molecular weight, and its antigen-binding site includes weight chain variable Area and light chain variable district.Though the molecular weight of the small molecular antibody is little but maintains the affinity of parent's monoclonal antibody, with parent's list Resist the same specificity.The species of the small molecular antibody mainly includes Fab antibody, Fv antibody, single-chain antibody (scFv) etc.. Fab antibody is by complete light chain (variable region V_LWith constant region C_L) and heavy chain Fd sections (variable region V_HWith the first constant region C_H1) pass through Disulfide bond is formed.Fv antibody is only connected by non-covalent bond by the variable region of light chain and heavy chain, is that antibody molecule has retained The minimum function fragment of whole antigen-binding site.Single-chain antibody (scFv) is that weight chain variable district and light chain variable district pass through connection sheet The single protein peptide chain molecule that section is formed by connecting.

Connected by junction fragment 1 between first functional domain and second functional domain, second functional domain and Connected by junction fragment 2 between 3rd functional domain.The present invention does not have particular/special requirement for the order of connection, as long as not limiting The purpose of the present invention.For example, it may be the C-terminal of first functional domain is connected with the N-terminal of second functional domain； The C-terminal of second functional domain is connected with the N-terminal of the 3rd functional domain.The present invention is for junction fragment 1 and connection sheet Section 2 does not have special restriction yet, as long as do not limit the purpose of the present invention.

Further, the junction fragment 1 and junction fragment 2 are selected from the junction fragment or immune globulin in units of G4S The hinge region fragment of white IgD.

The G4S is specially GGGGS.The junction fragment in units of G4S includes one or more G4S units.Example Such as, the G4S units for being, two, three or more than four can be included.In some embodiments of the present invention, one is listed In the bifunctional molecule of monomeric form, connected by the junction fragment 1 in units of G4S between the first functional domain and the second functional domain Connect, connected by the junction fragment 2 in units of G4S between the second functional domain and the 3rd functional domain.The junction fragment 1 contains One G4S unit, the aminoacid sequence of the junction fragment is as shown in SEQ ID NO.23.The junction fragment 2 contains three G4S units, the aminoacid sequence of the junction fragment is as shown in SEQ ID NO.25.

The hinge region fragment of the Immunoglobulin IgD can be the hinge Ala90-Val170 of Immunoglobulin IgD.This In some embodiments of invention, list in the bifunctional molecule of a dimeric forms, the first functional domain and the second functional domain it Between connected by the junction fragment 1 in units of G4S, between the second functional domain and the 3rd functional domain pass through Immunoglobulin IgD The connection of hinge region fragment, the hinge Ala90- of the hinge region fragment of the Immunoglobulin IgD for Immunoglobulin IgD Val170.The junction fragment 1 contains a G4S unit, the aminoacid sequence such as SEQ ID NO.27 institutes of the junction fragment Show.The aminoacid sequence of the junction fragment 2 is as shown in SEQ ID NO.29.The junction fragment 2 can be mutual by disulfide bond Connection forms dimer.

In the preferred embodiment, the structural representation of three functional molecular is as shown in Figure 1.Three function It can also be dimeric forms that molecule can be monomeric form.The structural representation of three functional moleculars of the monomeric form of the present invention As shown in Figure 1A, the first functional domain containing with CD19 antigen bindings in the structure of three functional molecular, one and CD3 Second functional domain of antigen binding, one is anti-with CD19 with the 3rd functional domain of CD28 antigen bindings, first functional domain The former single-chain antibody (scFv) for combining, second functional domain is and the single-chain antibody of CD3 antigen bindings (scFv), the described 3rd Functional domain is and the single-chain antibody of CD28 antigen bindings (scFv).The structure of three functional moleculars of the dimeric forms of the present invention is shown It is intended to as shown in Figure 1B, the first functional domain containing two with CD19 antigen bindings in the structure of three functional molecular, two With the second functional domain of CD3 antigen bindings, two with the 3rd functional domain of CD28 antigen bindings, first functional domain be with The single-chain antibody (scFv) of CD19 antigen bindings, second functional domain is and the single-chain antibody of CD3 antigen bindings (scFv), institute Stating the 3rd functional domain is and the single-chain antibody of CD28 antigen bindings (scFv).Three functional moleculars of the dimeric forms of the present invention Antigen binding potency is two times of monomeric form.Due to the first signal of T cell activation (CD3) and secondary signal (CD28) plus Times, cause T cell activation more abundant, it is higher to the fragmentation effect of target cell；The doubling of CD19 single-chain antibody domains makes which Identification to target cell is also more accurate, therefore dimer has more preferable using effect compared with monomer.

Specifically, first functional domain is the single-chain antibody of anti-CD19.The single-chain antibody of the anti-CD19 includes heavy chain Variable region and light chain variable district.The aminoacid sequence of the weight chain variable district of the single-chain antibody of the anti-CD19 such as SEQ ID NO.6 It is shown.The aminoacid sequence of the light chain variable district of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.7.Further, The aminoacid sequence of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.5.

Single-chain antibody of second functional domain for AntiCD3 McAb.The single-chain antibody of the AntiCD3 McAb includes weight chain variable district and light Chain variable region.The aminoacid sequence of the weight chain variable district of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.9.It is described anti- The aminoacid sequence of the light chain variable district of the single-chain antibody of CD3 is as shown in SEQ ID NO.10.Further, the AntiCD3 McAb The aminoacid sequence of single-chain antibody is as shown in SEQ ID NO.8.

3rd functional domain is the single-chain antibody of anti-CD28.The single-chain antibody of the anti-CD28 include weight chain variable district and Light chain variable district.The aminoacid sequence of the weight chain variable district of the single-chain antibody of the anti-CD28 is as shown in SEQ ID NO.12.Institute The aminoacid sequence of light chain variable district of the single-chain antibody of anti-CD28 is stated as shown in SEQ ID NO.13.The anti-CD28's is single-stranded The aminoacid sequence of antibody is as shown in SEQ ID NO.11.

In the preferable case of this case, the aminoacid sequence of three functional moleculars of monomeric form is as shown in SEQ ID NO.1. The aminoacid sequence of three functional moleculars of dimeric forms is as shown in SEQ ID NO.3.But it is not limited in preferably case of the invention Cited concrete form.

3rd, the polynucleotide of three functional moleculars are encoded

The polynucleotide of coding three functional molecular of the present invention, can be DNA form or rna form.DNA form bag Include the DNA of cDNA, genomic DNA or synthetic.DNA can be single-stranded or double-strand.

The polynucleotide of coding three functional molecular of the present invention, can pass through well known to those skilled in the art any It is prepared by appropriate technology.The technology sees the general description of this area, such as《Molecular Cloning:A Laboratory guide》(J. Pehanorm Brookers Deng, Science Press, 1995).The including but not limited to method such as recombinant DNA technology, chemosynthesis；For example with overlap extension PCR methods.

In the preferred embodiment, encode the nucleotides sequence of the weight chain variable district of the single-chain antibody of the anti-CD19 Row are as shown in SEQ ID NO.15.

The nucleotide sequence of light chain variable district of the single-chain antibody of the anti-CD19 is encoded as shown in SEQ ID NO.16.

The nucleotide sequence of single-chain antibody of the anti-CD19 is encoded as shown in SEQ ID NO.14.

The nucleotide sequence of weight chain variable district of the single-chain antibody of the AntiCD3 McAb is encoded as shown in SEQ ID NO.18.

The nucleotide sequence of light chain variable district of the single-chain antibody of the AntiCD3 McAb is encoded as shown in SEQ ID NO.19.

The nucleotide sequence of single-chain antibody of the AntiCD3 McAb is encoded as shown in SEQ ID NO.17.

The nucleotide sequence of weight chain variable district of the single-chain antibody of the anti-CD28 is encoded as shown in SEQ ID NO.21.

The nucleotide sequence of light chain variable district of the single-chain antibody of the anti-CD28 is encoded as shown in SEQ ID NO.22.

The nucleotide sequence of single-chain antibody of the anti-CD28 is encoded as shown in SEQ ID NO.20.

The nucleotide sequence such as SEQ ID NO.24 of junction fragment 1 of the encoding amino acid sequence as shown in SEQ ID NO.23 It is shown.

The nucleotide sequence such as SEQ ID NO.26 of junction fragment 2 of the encoding amino acid sequence as shown in SEQ ID NO.25 It is shown.

The nucleotide sequence such as SEQ ID NO.28 of junction fragment 1 of the encoding amino acid sequence as shown in SEQ ID NO.27 It is shown.

The nucleotide sequence such as SEQ ID NO.30 of junction fragment 2 of the encoding amino acid sequence as shown in SEQ ID NO.29 It is shown.

Further, the nucleotide sequence of three functional moleculars of coded cell form is as shown in SEQ ID NO.2.Coding two The nucleotide sequence of three functional moleculars of dimer form is as shown in SEQ ID NO.4.

4th, expression vector

The expression vector of the present invention contains the polynucleotide for encoding three functional molecular.Those skilled in the art Well known method can be used for building the expression vector.These methods include recombinant DNA technology, DNA synthetic technologys etc..To can compile The DNA of the code fusion protein is effectively connected in the multiple clone site in carrier, to instruct mRNA synthesis and then expressing protein, Or it is used for homologous recombination.In the preferable case of the present invention, the expression vector adopts pcDNA3.1.The host cell is adopted Chinese hamster ovary cell (Chinese hamster ovary ce1l, CHO).

5th, the method for preparing three functional moleculars

The method for preparing aforementioned three functional molecular of the present invention, including：Build the table containing three functional molecular gene orders Up to carrier, the expression vector containing three functional molecular gene orders is converted into into host cell abduction delivering then, produced from expression Separate in thing and obtain three described functional moleculars.In the preferable case of the present invention, the expression vector adopts pcDNA3.1.It is described Host cell adopts Chinese hamster ovary cell (Chinese hamster ovary ce1l, CHO).

6th, the purposes of three functional moleculars

Three functional moleculars of the present invention can be used for anti-tumor medicine.It is the swollen of the CD19 positives that the tumor is cell surface Tumor.

In present pre-ferred embodiments, with human peripheral blood single nucleus cell (Peripheral blood mononuclear Cell, PBMC) for experiment material, with three functional moleculars of the above-mentioned monomeric form prepared by the present invention, dimeric forms three The anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb) of functional molecular and purchase are respectively acting on same donor CIK cell (CD3 prepared by the human blood PBMC in source⁺CD56⁺) and CCL-86Raji lymphoma cell (CD19⁺).As a result find, After three functional moleculars of the addition present invention, CIK cell increases significantly to the killing-efficiency of Raji cells, positive to CD19 The target killing activity of property tumor cell is superior to anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb).

7th, cancer therapeutics compositions

The cancer therapeutics compositions of the present invention, it is pharmaceutically acceptable containing aforementioned three functional molecular and at least one Carrier or excipient.It is the positive tumors of CD19 that the tumor is cell surface.

Pharmaceutical composition provided by the present invention can be present with various dosage forms, such as be used for the injection of intravenous injection etc., use In the transdermic absorbent of subcutaneous injection, epidermis external application etc., for spraying the spray of nose, larynx, oral cavity, epidermis, mucosa etc., for dripping The drop of nose, eye, ear etc., it is various for the suppository of anorectal etc., tablet, powder, granule, capsule, oral liquid, unguentum, cream etc. Form, and the compositionss of formulation for pulmonary delivery and other parenterai administrations.The medicine of above-mentioned various dosage forms can be according to pharmacy It is prepared by the conventional method in field.

The carrier include the conventional diluent of pharmaceutical field, excipient, filler, binding agent, wetting agent, disintegrating agent, Absorption enhancer, surfactant, absorption carrier, lubricant etc..The Pharmaceutical composition can also add flavouring agent, sweeting agent Deng.

Pharmaceutical preparation as above can be to mammal Clinical practice, including humans and animals, can with intravenous administration or The approach administrations such as person's mouth, nose, skin, lung suction.The preferred weekly dose of said medicine be 0.1-5mg/kg body weight, the preferred course for the treatment of For 10 to 30 days.Once daily, or divided doses.No matter which kind of medication is adopted, the optimal dose of individuals should basis Depending on specific treatment.

8th, the method for external treatment tumor

The method of the external treatment tumor of the present invention, including aforementioned three functional molecular or cancer therapeutics compositions are applied For in tumor patient.It is the positive tumors of CD19 that the tumor is cell surface.Methods described can be non-treatment purpose. In present pre-ferred embodiments, with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC it is) experiment material, with three functions point of three functional moleculars of the above-mentioned monomeric form prepared by the present invention, dimeric forms The anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb) of son and purchase are respectively acting on same donor source CIK cell (CD3 prepared by human blood PBMC⁺CD56⁺) and CCL-86Raji lymphoma cell (CD19⁺).As a result find, in addition After three functional moleculars of the present invention, CIK cell increases significantly to the killing-efficiency of Raji cells, to CD19 positive tumors The target killing activity of cell is superior to anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb).

The present invention is directed to the deficiency of the CAR-T technologies of anti-CD19/ AntiCD3 McAbs BiTE bi-specific antibodys and targeting CD19, Being constructed by the method for genetic engineering and antibody engineering can be while recognizes three functional moleculars of CD19, CD3 and CD28.The molecule There is in terms of preparation technology and practical application obvious advantage：While imparting T cell is positive cell targeted to CD19 Further increase effect of activating T cell, the T cell mediated when individually adding to the combination of CD19 positive target cells with kill Hinder effect and be superior to anti-CD19/ AntiCD3 McAbs BiTE bi-specific antibodys, be better than the CAR-T of targeting CD19 in the convenience for using Technology.

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific concrete in order to describe Embodiment, rather than in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, Generally according to normal condition, or according to the condition proposed by each manufacturer.

When embodiment provides numerical range, it should be appreciated that except non-invention is otherwise noted, two ends of each numerical range Between point and two end points, any one numerical value can select.Unless otherwise defined, the present invention used in all technologies and The same meaning that scientific terminology is generally understood that with those skilled in the art of the present technique.Except the concrete grammar used in embodiment, equipment, Outside material, the record of grasp and the present invention according to those skilled in the art to prior art can also be used and this Any method of the similar or equivalent prior art of method, equipment described in inventive embodiments, material, equipment and material come real The existing present invention.

Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using this technology lead The conventional molecular biology in domain, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniquess of association area.The perfect explanation in existing document of these technologies, specifically can be found in Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The structure of 1 CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D carrier for expression of eukaryon of embodiment

In the present invention, with mankind's CD19 albumen on lymphoma B cell surface, T cell surface mankind's CD3 and CD28 albumen TiTE three-specific antibodies for target spot are named as CD19-CD3-CD28TsAb.

First, CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D constructing plans design

The concrete constructing plans of CD19-CD3-CD28TsAb_M of monomeric form are：Anti- CD19scFv, AntiCD3 McAb scFv and anti- The sequence of CD28scFv is connected by junction fragment (Linker), specifically, by connecting between anti-CD19scFv and AntiCD3 McAb scFv Tab segments 1 (Linker 1) are connected, and then pass through junction fragment 2 (Linker 2) between AntiCD3 McAb scFv and anti-CD28scFv sequences It is connected.

The concrete constructing plans of CD19-CD3-CD28TsAb_D of dimeric forms are：Anti- CD19scFv, AntiCD3 McAb scFv and The sequence of anti-CD28scFv is connected by junction fragment (Linker), specifically, is passed through between anti-CD19scFv and AntiCD3 McAb scFv Junction fragment 1 (Linker 1) is connected, with IgD hinge regions (Ala90- between AntiCD3 McAb scFv and anti-CD28scFv sequences Val170) it is connected as junction fragment 2 (Linker 2).

To make three-specific antibody be expressed in mammalian cell, for anti-CD19scFv, AntiCD3 McAb scFv, resist CD28scFv sequences have carried out the codon optimization of suckling system expression.

Specifically, the nucleotide sequence of the weight chain variable district of anti-CD19scFv is as shown in SEQ ID NO.15, specially：

CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGG CTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCT GGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGC ACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCAC CGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable district of anti-CD19scFv as shown in SEQ ID NO.16, specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAG CCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGA TCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTG AACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGG CGGCGGCACCAAGCTGGAGATCAAG。

The nucleotide sequence of anti-CD19scFv as shown in SEQ ID NO.14, specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAG CCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGA TCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTG AACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGG CGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGC AGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCC TTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGG CGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCT ACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGC CGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

The nucleotide sequence of the weight chain variable district of AntiCD3 McAb scFv as shown in SEQ ID NO.18, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGG CTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCA ACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGC ACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCA CTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable district of AntiCD3 McAb scFv as shown in SEQ ID NO.19, specially：

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAG CAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCA AGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATG GAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCT GGAGCTGAAG。

The nucleotide sequence of AntiCD3 McAb scFv as shown in SEQ ID NO.17, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGG CTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCA ACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGC ACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCA CTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCG GCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAG AAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCC CAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCA GCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCC CTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

The nucleotide sequence of the weight chain variable district of anti-CD28scFv as shown in SEQ ID NO.21, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGG CTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCT ACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGC ACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCT GGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable district of anti-CD28scFv as shown in SEQ ID NO.22, specially：

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAG CCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCA GCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAA GGTGGAGATCAAGCGC。

The nucleotide sequence of anti-CD28scFv as shown in SEQ ID NO.20, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGG CTACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCT ACCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGC ACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCT GGACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCG GCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTG ACCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAA GCTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACT TCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTAC ACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence such as SEQ ID of the CD19-CD3-CD28TsAb_M junction fragments 1 (Linker 1) of monomeric form Shown in NO.24, specially：

GGTGGCGGAGGGTCC。

The nucleotide sequence such as SEQ ID of the CD19-CD3-CD28TsAb_M junction fragments 2 (Linker 2) of monomeric form Shown in NO.26, specially：

GGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGT。

The nucleotide sequence such as SEQ of the CD19-CD3-CD28TsAb_D junction fragments 1 (Linker 1) of dimeric forms Shown in ID NO.28, specially：

GGTGGCGGAGGGTCC。

The nucleotide sequence such as SEQ of the CD19-CD3-CD28TsAb_D junction fragments 2 (Linker 2) of dimeric forms Shown in ID NO.30, specially：

GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCA GCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGG AGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAG CCCCTGGGCGTG。

Antibody-secreting be have selected to make three-specific antibody that simultaneously successful secretion is expressed in CHO-S cells in culture medium The signal peptide of type expression is used for this embodiment.

The aminoacid sequence of the secreting, expressing signal peptide as shown in SEQ ID NO.31, specially：

MTRLTVLALLAGLLASSRA。

The nucleotide sequence of the secreting, expressing signal peptide as shown in SEQ ID NO.32, specially：

ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGG

CC。

2nd, CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention, from mammalian cell albumen transient expression vector pcDNA3.1 (being purchased from Shanghai Ying Jun bio tech ltd).To build the three-specific antibody of monomer and dimeric forms, separately design Primer as shown in table 1, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template needed for amplification is by reviving Zhou Hongxun Science and Technology Ltd.s synthesize.

Build for the clone of CD19-CD3-CD28TsAb_M, expand first by primer pcDNA3.1-Sig-F and Sig-R Increase and signal fragments of peptides, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3- (GGGGS)₃- CD28-F and pcDNA3.1-CD28-R amplify anti-CD19scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, (GGGGS)₃The gene order of the anti-CD28scFv of Linker 2+；Build for the clone of CD19-CD3-CD28TsAb_D, equally Signal fragments of peptides is amplified first by primer pcDNA3.1-Sig-F and Sig-R, primer Sig-CD19-F is then utilized respectively Expand with CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD28-F and pcDNA3.1-CD28-R Increase the gene sequence for anti-CD19scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge region Linker 2, anti-CD28scFv Row.After amplification is finished, utilizeMono- step directed cloning test kits of PCR are (limited purchased from Wujiang offshore protein science and technology Company) splice respectively monomer and dimeric forms three-specific antibody full-length gene order and it is seamless be cloned into Jing EcoRI and On the pcDNA3.1 expression vectors of HindIII linearization process, bacillus coli DH 5 alpha is converted, positive gram is carried out using bacterium colony PCR Grand identification, the recon (recombiant plasmid) for being accredited as the positive carry out sequencing identification.Correct recon (restructuring matter will be subsequently sequenced Grain) arrange plasmid in take out, for the transfection of CHO-S cells.

Jing is sequenced to be known, the CD19-CD3- of the CD19-CD3-CD28TsAb and dimeric forms of monomeric form The full-length gene order of CD28TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-CD28TsAb_M of monomeric form has as shown in SEQ ID NO.2 Body is：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAG CCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGA TCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTG AACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGG CGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGC AGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCC TTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGG CGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCT ACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGC CGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACAT CAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACA CCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCC AGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGC CTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACT GCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGC GGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGT GACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGC GCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTAC AGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGAC CTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTC AGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGC TACACCTTCACCAGCTACTACATCCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTA CCCCGGCAACGTGAACACCAACTACAACGAGAAGTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCA CCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTG GACTGGAACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGG CAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGA CCATCACCTGCCACGCCAGCCAGAACATCTACGTGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAG CTGCTGATCTACAAGGCCAGCAACCTGCACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTT CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACA CCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of the CD19-CD3-CD28TsAb_D of dimeric forms as shown in SEQ ID NO.4, specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAG CCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGA TCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTG AACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGG CGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGC AGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCC TTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGG CGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCT ACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGC CGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACAT CAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACA CCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCC AGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGC CTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACT GCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGC GGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGT GACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGC GCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTAC AGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGAC CTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCA AGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCC ACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGAC CAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA AGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGGTG CGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCTGCATCTACCCCGGCAACGTGAACACCAACTACAACGAGAA GTTCAAGGACCGCGCCACCCTGACCGTGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCG ACGACACCGCCGTGTACTTCTGCACCCGCAGCCACTACGGCCTGGACTGGAACTTCGACGTGTGGGGCCAGGGCACC ACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGAC CCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCACGCCAGCCAGAACATCTACG TGTGGCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAACCTGCACACC GGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGGCCAGACCTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGC GC。

Primer used in 1. three-specific antibody gene cloning of table

The expression and purification one, CD19- of embodiment 2 CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D The expression of CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D

1.1.CHO-S cell (being purchased from Thermo Fisher Scientific companies) is transfected and passes within first 1 day density for 0.5 ～0.6 × 10⁶/ml；

1.2. transfect the same day carry out cell density statistics, when density be 1～1.4 × 10⁶/ ml, vigor>When 90%, can use In plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D) Two centrifuge tube/culture bottles need to be prepared, by taking 20ml as an example, placed respectively, prepared recombiant plasmid in Example 1：

1. 600 μ l PBS of middle addition are managed, 20 μ g recombiant plasmid are mixed；

Manage 2. middle addition 600 μ l PBS, 20ul FreeStyle^TMMAX Transfection Reagent (are purchased from Thermo Fisher Scientific companies), mix；

1.4., by the transfection reagent after dilution, in adding the recombiant plasmid to dilution, mix homogeneously is configured to transfection multiple Compound；

1.5. after transfection composite stands 15～20min, during single drop at the uniform velocity adds cell culture；

1.6. in 37 DEG C, CO₂Concentration 8%, carries out Transfected cells culture under the conditions of shaking speed 130rpm, receive after 5 days Collection culture supernatant carries out destination protein detection of expression.

2nd, the purification of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, adds buffer 20mM PB, 200mM NaCl to adjust pH to 7.5；

2.2 Protein L affinity chromatograph column purifications

Protein purification chromatographic column：Protein L affinity columns (are purchased from GE Healthcare companies, column volume 1.0ml)

Buffer A (Buffer A)：PBS, pH7.4

Buffer B (Buffer B)：0.1M Glycine,pH3.0

Buffer C (Buffer C)：0.1M Glycine,pH2.7

Purge process：Using 100 type protein purification systems of AKTA explorer (being purchased from GE Healthcare companies), With Buffer A pretreatment Protein L affinity columns, culture supernatant loading is taken, collect effluent.After loading is finished, with extremely Few 1.5ml Buffer A balance chromatographic columns, use Buffer B and Buffer C eluting respectively, collect destination protein and wash after balance (collecting pipe of eluent needs the 1M Tris for being previously added 1%, and pH8.0 is neutralizing eluent pH value, Tris final concentrations for de- liquid About 10mM), finally concentration dialysis is into buffer PBS.

CD19-CD3-CD28TsAb_M the and CD19-CD3-CD28TsAb_D recombiant protein Jing SDS-PAGE of final purification Analysis, under reduction and non reducing conditions, electrophoretogram is as shown in Figure 2.It can be seen that Jing Protein L affinity columns are pure After change, the purity of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D recombiant proteins is equal>95%：Wherein CD19- The theoretical molecular of CD3-CD28TsAb_M recombiant proteins is 81.3kDa, and under reduction and non reducing conditions, the albumen is presented list One electrophoretic band, molecular weight are consistent with monomer, therefore the three-specific antibody is monomeric form (Fig. 2A)；CD19-CD3- The theoretical molecular of CD28TsAb_D recombiant proteins be 89.1kDa, the presented molecular weight of protein electrophoresises band under reducing condition It is consistent with monomer, the presented molecular weight of electrophoretic band (～180kDa) consistent with dimer (Fig. 2 B), explanation under non reducing conditions Two protein moleculars can form disulfide bond by IgD hinge regions and be connected with each other, therefore the three-specific antibody is dimeric forms.

Additionally, the recombiant protein sample Jing N/C terminal sequence analysis of purification, as a result show expressed recombiant protein sample Frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analyses further confirm that CD19-CD3-CD28TsAb_M For monomeric form, CD19-CD3-CD28TsAb_D is dimeric forms.

Therefore, can learn, the aminoacid sequence such as SEQ ID NO.1 institutes of the CD19-CD3-CD28TsAb_M of monomeric form Show, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTL NIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYA FSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVG RYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINP SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGS GGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY SLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGL DWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPK LLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

The aminoacid sequence of the CD19-CD3-CD28TsAb_D of dimeric forms as shown in SEQ ID NO.3, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTL NIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYA FSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVG RYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINP SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGS GGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY SLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPA TTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWV RQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGT TVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

The aminoacid sequence of anti-CD19scFv as shown in SEQ ID NO.5, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTL NIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYA FSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVG RYYYAMDYWGQGTTVTVSS。

The aminoacid sequence of the weight chain variable district of anti-CD19scFv as shown in SEQ ID NO.6, specially：

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSS TAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

The aminoacid sequence of the weight chain variable district of anti-CD19scFv as shown in SEQ ID NO.7, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTL NIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK。

The aminoacid sequence of AntiCD3 McAb scFv as shown in SEQ ID NO.8, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS TAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGE KVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNP LTFGAGTKLELK。

The aminoacid sequence of the weight chain variable district of AntiCD3 McAb scFv as shown in SEQ ID NO.9, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS TAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。

The aminoacid sequence of the light chain variable district of AntiCD3 McAb scFv as shown in SEQ ID NO.10, specially：

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSM EAEDAATYYCQQWSSNPLTFGAGTKLELK。

The aminoacid sequence of anti-CD28scFv as shown in SEQ ID NO.11, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSIS TAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRV TITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPY TFGGGTKVEIKR。

The aminoacid sequence of the weight chain variable district of anti-CD28scFv as shown in SEQ ID NO.12, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSIS TAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS。

The aminoacid sequence of the light chain variable district of anti-CD28scFv as shown in SEQ ID NO.13, specially：

DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQGQTYPYTFGGGTKVEIKR。

The aminoacid sequence such as SEQ of junction fragment 1 (Linker 1) in the CD19-CD3-CD28TsAb_M of monomeric form Shown in ID NO.23, specially：GGGGS.

The aminoacid sequence such as SEQ of junction fragment 2 (Linker 2) in the CD19-CD3-CD28TsAb_M of monomeric form Shown in ID NO.25, specially：GGGGSGGGGSGGGGS.

The aminoacid sequence such as SEQ of junction fragment 1 (Linker 1) in the CD19-CD3-CD28TsAb_D of dimeric forms Shown in ID NO.27, specially：GGGGS.

The aminoacid sequence such as SEQ of junction fragment 2 (Linker 2) in the CD19-CD3-CD28TsAb_D of dimeric forms Shown in ID NO.29, specially：

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQ PLGV。

Embodiment 3：ELISA detects the antigen of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D

Binding activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind's CD28-hFc fusion protein (are purchased from Wu Jiang Jinan protein Science and Technology Ltd.) 96 orifice plates are coated with respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes, Coating condition be 37 DEG C of 1 hour or 4 DEG C overnight, the formula for being coated with buffer (PBS) is：3.58g Na₂HPO₄, 0.24g NaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, adjusts pH to 7.4 with 1mol/L HCl or 1mol/L NaOH, and moisturizing is extremely 1L；

2. close：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added.37 DEG C of closings 1 hour；

3. it is loaded：After PBS board-washings 4 times, the three-specific antibody sample of purification, 100 μ l/ holes, 37 DEG C of incubations 1 are separately added into Hour, sample gradient compound method：With the CD19-CD3-CD28TsAb_M or CD19-CD3-CD28TsAb_D of 10 μ g/ml purification As initial concentration, 6 gradients of doubling dilution are carried out, each gradient arranges 2 multiple holes；

4. develop the color：It is after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 times, dilute by 1/5000 with confining liquid PBSA Release the anti-His of HRP labellings₆Fusion tag antibody (is purchased from Abcam companies), adds by 100 μ l/ holes, and 37 DEG C are incubated 1 hour.PBS After board-washing 4 times, addition nitrite ion TMB (is purchased from KPL companies), and 100 μ l/ holes, room temperature lucifuge are developed the color 5～10 minutes；

5. terminating reaction is determined with result：Addition terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate reader Lower reading light absorption value (OD450).

ELISA results are as shown in Figure 3 A and Figure 3 B：Fig. 3 A illustrate CD19-CD3-CD28TsAb_M and recombinant antigen CD19- HFc, CD3-hFc and CD28-hFc are respectively provided with external binding activity, wherein CD28 binding activity activity highest, CD19 binding activity Take second place, CD3 binding activity is weaker；Fig. 3 B illustrate CD19-CD3-CD28TsAb_D and recombinant antigen CD19-hFc, CD3-hFc and CD28-hFc equally has external binding activity, wherein CD28 binding activity highest, and CD19 binding activity is taken second place, and CD3 is combined and lived Property is weaker.

Embodiment 4：The cell linking experiment of three specific antibodies and Mediated by Bi-specific Antibodies

Using CCL-86Raji lymphoma cells (being purchased from ATCC) as the positive target cells of CD19, TIB-152Jurkat is thin , used as the positive effector lymphocytes of CD3 and CD28, the TiTE tri- of comparison monomeric form of the present invention is special anti-for born of the same parents' (being purchased from ATCC) Body (CD19-CD3-CD28TsAb_M), the TiTE three-specific antibodies (CD19-CD3-CD28TsAb_D) of dimeric forms and What anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) mediated Cell is connected activity difference.

Cell is connected experimental procedure：

1st, Raji cell～1 × 10 are taken⁵It is individual, high, medium and low 3 experimental grouies of concentration are set, add final concentration of 45 respectively, 0.45th, CD19-CD3BsAb, CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D of 0.0045ng/ml, stands 5min；To be not added with the cell of any antibody as blank；

2nd, the Jurkat cell of equal number is taken, is respectively added in above-mentioned Raji cell samples, 37 DEG C of incubators are placed 1h, takes out cell and softly rocks 30s, stand 2min, and the agglomerating situation of basis of microscopic observation cell is simultaneously taken pictures；

As a result it is as shown in Figure 4：Under conditions of any antibody is not added with, Raji cells are agglomerating without assembling with Jurkat cell (Fig. 4 A), illustrates there is no non-specific linking between two kinds of cells；Under conditions of addition high concentration antibody (45ng/ml), 3 Raji cells in individual experimental group with Jurkat cell substantially agglomerating (Fig. 4 B-D), illustrate that the TiTE tri- of two kinds of forms is special anti- Body (CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies (CD19-CD3BsAb) cell linking activity is more or less the same at higher concentrations；The concentration antibody (0.45ng/ml) in addition Under the conditions of, it is bright with Jurkat cell that CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D can still result in Raji cells Aobvious agglomerating, CD19-CD3BsAb can cause two kinds of cells a small amount of agglomerating (Fig. 4 E-G), illustrate that the TiTE tri- of two kinds of forms is special anti- Body cell linking activity under intermediate concentration is superior to BiTE bi-specific antibodys；In addition low concentration antibody (0.0045ng/ Ml, under conditions of), it is substantially agglomerating with Jurkat cell that CD19-CD3-CD28TsAb_D can still result in Raji cells, CD19-CD3- CD28TsAb_M can cause two kinds of cells agglomerating on a small quantity, and CD19-CD3BsAb cannot make cell agglomerating (Fig. 4 H-J), illustrate two Cell linking activity resists the TiTE three-specific antibodies of dimer form better than the TiTE tri-specifics of monomeric form at low concentrations Body, and BiTE bi-specific antibodys at low concentrations it is acellular linking activity.

Embodiment 5：The cell killing experiment of three specific antibodies and Mediated by Bi-specific Antibodies

With human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) for experiment material Material, with the tri- specific antibody CD19-CD3-CD28TsAb_M of TiTE of the above-mentioned monomeric form prepared by the present invention, dimeric forms Tri- specific antibody CD19-CD3-CD28TsAb_D of TiTE and purchase anti-CD19/ AntiCD3 McAbs BiTE bispecific antibody (CD19- CD3BsAb) it is respectively acting on CIK cell (CD3 prepared by the human blood PBMC of same donor source⁺CD56⁺) drench with CCL-86Raji Bar oncocyte (CD19⁺), cell death situation is detected, compares three kinds of antibody-mediated CIK effector lymphocytes to CCL-86Raji targets The killing-efficiency difference of cell.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the new volunteer's anticoagulated blood for extracting, add isopyknic medical saline, along from Heart tube wall is slowly added to and the isopyknic lymphocyte separation medium of blood (purchased from GE Healthcare companies), keeps liquid level point Substantially, 2000rpm centrifugation 20min draw the cellular layer of middle white haze shape in new centrifuge tube, add 2 times with upper volume layer PBS washing, 1100rpm centrifugation 10min, repeated washing once, with 15 serum-free cultures of X-vivo of a small amount of pre-cooling Base (being purchased from Lonza companies) is resuspended, and cell counting is stand-by；

2.CIK cell culture and amplification：PBMC (is purchased from CIK basal mediums (90%X-vivo15+10%FBS) Gbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml, is added to full length antibody Anti-CD3 (5ug/ml), total length and resists In body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody with NovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), while addition cytokine IFN-γ (200ng/ml, Purchased from Wujiang Alongshore Protein Technology Co., Ltd.) and IL-1 β (2ng/ml, purchased from the limited public affairs of Wujiang offshore protein science and technology Department), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, add 500U/ The IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues culture, per counting within 2-3 days and with adding 500U/ml The CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out passage；

Killing-efficiency of the 3.CIK cells to Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is 100uL, takes the CIK cell 1 × 10 of above-mentioned culture⁵It is individual, add Raji cells 5 × 10⁵Individual (CIK effector lymphocyte：Raji target cells (E：T ratios) for 1：5) or 1 × 10⁵Individual (E:T compares 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3BsAb, CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D antibody samples, room temperature mix 3- 5min, after 37 DEG C co-culture 3h, adds the CCK8 of 10uL per hole, and 37 degree are continued reaction 2-3h, subsequently survey OD with microplate reader₄₅₀Value, Cell killing efficiency, per group of experiment duplicate detection 3 times are calculated according to below equation；Killed with the cell for being not added with any antibody simultaneously Hinder efficiency as blank.

As a result it is as shown in Figure 5：As CIK effector lymphocyte：Raji target cell (E：T ratios) it is respectively 1：5 and 1：When 1, do not adding Plus under conditions of any antibody, 3h cell killings efficiency is about 17% (Fig. 5 A) and 21% (Fig. 5 B)；Resist in addition higher concentration Under conditions of body (25,12.5,6.25ng/ml), CIK cell is significantly increased to the killing-efficiency of Raji cells, wherein Cell killing efficacy that CD19-CD3-CD28TsAb_D is mediated is best, work as E：T ratios are 1：When 5, killing-efficiency respectively may be about 36%th, 29% and 30%, work as E：T ratios are 1：When 1, killing-efficiency respectively may be about 85%, 90% and 85%, CD19-CD3- The effect of CD28TsAb_M takes second place, works as E：T ratios are 1：When 5, killing-efficiency respectively may be about 30%, 23% and 26%, work as E：T ratios are 1：When 1, the effect of killing-efficiency about 86%, 82% and 81%, CD19-CD3BsAb is most weak, work as E：T ratios are 1：When 5, kill Efficiency respectively may be about 23%, 22% and 22%, work as E：T ratios are 1：When 1, killing-efficiency respectively may be about 80%, 55% and 56%； Under conditions of addition low concentration antibody (3.125ng/ml), CD19-CD3-CD28TsAb_D and CD19-CD3-CD28TsAb_M The CIK cell for being mediated still has raising to a certain extent, works as E to the killing-efficiency of Raji cells：T is 1：When 5, killing-efficiency point Not Yue Wei 23% and 22%, work as E：T ratios are 1：When 1, killing-efficiency respectively may be about 82% and 70%, and CD19-CD3BsAb with it is empty Substantially no effect is compared in white control, and the above results illustrate the T cell pair mediated by tri- specific antibodies of TiTE of two kinds of forms The target killing activity of CD19 positive tumor cells is superior to BiTE bi-specific antibodys, and wherein dimeric forms are compared with monomeric form With more preferable effect.

The above, only presently preferred embodiments of the present invention, not any formal and substantial to present invention restriction, It should be pointed out that for those skilled in the art, on the premise of without departing from the inventive method, can also make Some improvement and supplement, these improve and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when make using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations for developing, are the Equivalent embodiments of the present invention；Meanwhile, all substantial technologicals pair according to the present invention Change, modification and the differentiation of any equivalent variations that above-described embodiment is made, still falls within the scope of technical scheme It is interior.

SEQUENCE LISTING

<110>Shanghai offshore bio tech ltd

<120>Three functional moleculars of a kind of combination CD19, CD3 and CD28 and its application

<130> 163905

<160> 43

<170> PatentIn version 3.3

<210> 1

<211> 756

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 1

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

1 5 10 15

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

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

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

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

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

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

305 310 315 320

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

325 330 335

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

340 345 350

Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr

355 360 365

Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly

370 375 380

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

385 390 395 400

Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

405 410 415

Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

485 490 495

Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

500 505 510

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

515 520 525

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

530 535 540

Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

545 550 555 560

Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys

565 570 575

Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala

580 585 590

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

595 600 605

Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly

610 615 620

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro

645 650 655

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His

660 665 670

Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln Lys Pro

675 680 685

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr

690 695 700

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

705 710 715 720

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

725 730 735

Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val

740 745 750

Glu Ile Lys Arg

755

<210> 2

<211> 2268

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 2

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaagggaggc 1500

ggaggttccg gcggtggggg atcggggggt ggagggagtc aggtgcagct ggtgcagagc 1560

ggcgccgagg tgaagaagcc cggcgccagc gtgaaggtga gctgcaaggc cagcggctac 1620

accttcacca gctactacat ccactgggtg cgccaggccc ccggccaggg cctggagtgg 1680

atcggctgca tctaccccgg caacgtgaac accaactaca acgagaagtt caaggaccgc 1740

gccaccctga ccgtggacac cagcatcagc accgcctaca tggagctgag ccgcctgcgc 1800

agcgacgaca ccgccgtgta cttctgcacc cgcagccact acggcctgga ctggaacttc 1860

gacgtgtggg gccagggcac caccgtgacc gtgagcagcg gcggcggcgg cagcggcggc 1920

ggcggcagcg gcggcggcgg cagcgacatc cagatgaccc agagccccag cagcctgagc 1980

gccagcgtgg gcgaccgcgt gaccatcacc tgccacgcca gccagaacat ctacgtgtgg 2040

ctgaactggt accagcagaa gcccggcaag gcccccaagc tgctgatcta caaggccagc 2100

aacctgcaca ccggcgtgcc cagccgcttc agcggcagcg gcagcggcac cgacttcacc 2160

ctgaccatca gcagcctgca gcccgaggac ttcgccacct actactgcca gcagggccag 2220

acctacccct acaccttcgg cggcggcacc aaggtggaga tcaagcgc 2268

<210> 3

<211> 822

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 3

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

1 5 10 15

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

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

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

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

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

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

305 310 315 320

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

325 330 335

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

340 345 350

Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr

355 360 365

Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly

370 375 380

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

385 390 395 400

Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

405 410 415

Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

485 490 495

Leu Lys Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu Ser

500 505 510

Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Gln Pro Gln Ala Glu

515 520 525

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

530 535 540

Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln

545 550 555 560

Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro

565 570 575

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

580 585 590

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

595 600 605

Thr Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

610 615 620

Glu Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn

625 630 635 640

Glu Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser

645 650 655

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

660 665 670

Tyr Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val

675 680 685

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

690 695 700

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

705 710 715 720

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

725 730 735

Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln

740 745 750

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu

755 760 765

His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

770 775 780

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

785 790 795 800

Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr

805 810 815

Lys Val Glu Ile Lys Arg

820

<210> 4

<211> 2466

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 4

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag 780

cagagcggcg ccgagctggc ccgccccggc gccagcgtga agatgagctg caagaccagc 840

ggctacacct tcacccgcta caccatgcac tgggtgaagc agcgccccgg ccagggcctg 900

gagtggatcg gctacatcaa ccccagccgc ggctacacca actacaacca gaagttcaag 960

gacaaggcca ccctgaccac cgacaagagc agcagcaccg cctacatgca gctgagcagc 1020

ctgaccagcg aggacagcgc cgtgtactac tgcgcccgct actacgacga ccactactgc 1080

ctggactact ggggccaggg caccaccctg accgtgagca gcgtggaggg cggcagcggc 1140

ggcagcggcg gcagcggcgg cagcggcggc gtggacgaca tccagctgac ccagagcccc 1200

gccatcatga gcgccagccc cggcgagaag gtgaccatga cctgccgcgc cagcagcagc 1260

gtgagctaca tgaactggta ccagcagaag agcggcacca gccccaagcg ctggatctac 1320

gacaccagca aggtggccag cggcgtgccc taccgcttca gcggcagcgg cagcggcacc 1380

agctacagcc tgaccatcag cagcatggag gccgaggacg ccgccaccta ctactgccag 1440

cagtggagca gcaaccccct gaccttcggc gccggcacca agctggagct gaaggccagc 1500

aagagcaaga aggagatctt ccgctggccc gagagcccca aggcccaggc cagcagcgtg 1560

cccaccgccc agccccaggc cgagggcagc ctggccaagg ccaccaccgc ccccgccacc 1620

acccgcaaca ccggccgcgg cggcgaggag aagaagaagg agaaggagaa ggaggagcag 1680

gaggagcgcg agaccaagac ccccgagtgc cccagccaca cccagcccct gggcgtgcag 1740

gtgcagctgg tgcagagcgg cgccgaggtg aagaagcccg gcgccagcgt gaaggtgagc 1800

tgcaaggcca gcggctacac cttcaccagc tactacatcc actgggtgcg ccaggccccc 1860

ggccagggcc tggagtggat cggctgcatc taccccggca acgtgaacac caactacaac 1920

gagaagttca aggaccgcgc caccctgacc gtggacacca gcatcagcac cgcctacatg 1980

gagctgagcc gcctgcgcag cgacgacacc gccgtgtact tctgcacccg cagccactac 2040

ggcctggact ggaacttcga cgtgtggggc cagggcacca ccgtgaccgt gagcagcggc 2100

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 2160

agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccacgccagc 2220

cagaacatct acgtgtggct gaactggtac cagcagaagc ccggcaaggc ccccaagctg 2280

ctgatctaca aggccagcaa cctgcacacc ggcgtgccca gccgcttcag cggcagcggc 2340

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 2400

tactgccagc agggccagac ctacccctac accttcggcg gcggcaccaa ggtggagatc 2460

aagcgc 2466

<210> 5

<211> 250

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of anti-CD19 scFv

<400> 5

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

1 5 10 15

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

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

115 120 125

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

130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met

145 150 155 160

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

165 170 175

Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly

180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln

195 200 205

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

210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

245 250

<210> 6

<211> 124

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the weight chain variable district of anti-CD19 scFv

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 7

<211> 111

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the light chain variable district of anti-CD19 scFv

<400> 7

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

1 5 10 15

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

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 8

<211> 243

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of AntiCD3 McAb scFv

<400> 8

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

65 70 75 80

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

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

180 185 190

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

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys

<210> 9

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the weight chain variable district of AntiCD3 McAb scFv

<400> 9

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 10

<211> 106

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the light chain variable district of AntiCD3 McAb scFv

<400> 10

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

1 5 10 15

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

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

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

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 11

<211> 243

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of anti-CD28 scFv

<400> 11

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala

145 150 155 160

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

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu His Thr Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg

<210> 12

<211> 120

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the weight chain variable district of anti-CD28 scFv

<400> 12

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

1 5 10 15

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

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 108

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of the light chain variable district of anti-CD28 scFv

<400> 13

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 His Ala Ser Gln Asn Ile Tyr Val Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr 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 Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr

85 90 95

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

100 105

<210> 14

<211> 750

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CD19 scFv

<400> 14

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aagggcggcg gcggcagcgg cggcggcggc 360

agcggcggcg gcggcagcca ggtgcagctg cagcagagcg gcgccgagct ggtgcgcccc 420

ggcagcagcg tgaagatcag ctgcaaggcc agcggctacg ccttcagcag ctactggatg 480

aactgggtga agcagcgccc cggccagggc ctggagtgga tcggccagat ctggcccggc 540

gacggcgaca ccaactacaa cggcaagttc aagggcaagg ccaccctgac cgccgacgag 600

agcagcagca ccgcctacat gcagctgagc agcctggcca gcgaggacag cgccgtgtac 660

ttctgcgccc gccgcgagac caccaccgtg ggccgctact actacgccat ggactactgg 720

ggccagggca ccaccgtgac cgtgagcagc 750

<210> 15

<211> 372

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the weight chain variable district of anti-CD19 scFv

<400> 15

caggtgcagc tgcagcagag cggcgccgag ctggtgcgcc ccggcagcag cgtgaagatc 60

agctgcaagg ccagcggcta cgccttcagc agctactgga tgaactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggccag atctggcccg gcgacggcga caccaactac 180

aacggcaagt tcaagggcaa ggccaccctg accgccgacg agagcagcag caccgcctac 240

atgcagctga gcagcctggc cagcgaggac agcgccgtgt acttctgcgc ccgccgcgag 300

accaccaccg tgggccgcta ctactacgcc atggactact ggggccaggg caccaccgtg 360

accgtgagca gc 372

<210> 16

<211> 333

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable district of anti-CD19 scFv

<400> 16

gacatccagc tgacccagag ccccgccagc ctggccgtga gcctgggcca gcgcgccacc 60

atcagctgca aggccagcca gagcgtggac tacgacggcg acagctacct gaactggtac 120

cagcagatcc ccggccagcc ccccaagctg ctgatctacg acgccagcaa cctggtgagc 180

ggcatccccc cccgcttcag cggcagcggc agcggcaccg acttcaccct gaacatccac 240

cccgtggaga aggtggacgc cgccacctac cactgccagc agagcaccga ggacccctgg 300

accttcggcg gcggcaccaa gctggagatc aag 333

<210> 17

<211> 729

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of AntiCD3 McAb scFv

<400> 17

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagcgtg 360

gagggcggca gcggcggcag cggcggcagc ggcggcagcg gcggcgtgga cgacatccag 420

ctgacccaga gccccgccat catgagcgcc agccccggcg agaaggtgac catgacctgc 480

cgcgccagca gcagcgtgag ctacatgaac tggtaccagc agaagagcgg caccagcccc 540

aagcgctgga tctacgacac cagcaaggtg gccagcggcg tgccctaccg cttcagcggc 600

agcggcagcg gcaccagcta cagcctgacc atcagcagca tggaggccga ggacgccgcc 660

acctactact gccagcagtg gagcagcaac cccctgacct tcggcgccgg caccaagctg 720

gagctgaag 729

<210> 18

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the weight chain variable district of AntiCD3 McAb scFv

<400> 18

gacatcaagc tgcagcagag cggcgccgag ctggcccgcc ccggcgccag cgtgaagatg 60

agctgcaaga ccagcggcta caccttcacc cgctacacca tgcactgggt gaagcagcgc 120

cccggccagg gcctggagtg gatcggctac atcaacccca gccgcggcta caccaactac 180

aaccagaagt tcaaggacaa ggccaccctg accaccgaca agagcagcag caccgcctac 240

atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc ccgctactac 300

gacgaccact actgcctgga ctactggggc cagggcacca ccctgaccgt gagcagc 357

<210> 19

<211> 318

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable district of AntiCD3 McAb scFv

<400> 19

gacatccagc tgacccagag ccccgccatc atgagcgcca gccccggcga gaaggtgacc 60

atgacctgcc gcgccagcag cagcgtgagc tacatgaact ggtaccagca gaagagcggc 120

accagcccca agcgctggat ctacgacacc agcaaggtgg ccagcggcgt gccctaccgc 180

ttcagcggca gcggcagcgg caccagctac agcctgacca tcagcagcat ggaggccgag 240

gacgccgcca cctactactg ccagcagtgg agcagcaacc ccctgacctt cggcgccggc 300

accaagctgg agctgaag 318

<210> 20

<211> 729

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CD28 scFv

<400> 20

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180

aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300

tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgacat ccagatgacc 420

cagagcccca gcagcctgag cgccagcgtg ggcgaccgcg tgaccatcac ctgccacgcc 480

agccagaaca tctacgtgtg gctgaactgg taccagcaga agcccggcaa ggcccccaag 540

ctgctgatct acaaggccag caacctgcac accggcgtgc ccagccgctt cagcggcagc 600

ggcagcggca ccgacttcac cctgaccatc agcagcctgc agcccgagga cttcgccacc 660

tactactgcc agcagggcca gacctacccc tacaccttcg gcggcggcac caaggtggag 720

atcaagcgc 729

<210> 21

<211> 360

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the weight chain variable district of anti-CD28 scFv

<400> 21

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctactaca tccactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatcggctgc atctaccccg gcaacgtgaa caccaactac 180

aacgagaagt tcaaggaccg cgccaccctg accgtggaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt acttctgcac ccgcagccac 300

tacggcctgg actggaactt cgacgtgtgg ggccagggca ccaccgtgac cgtgagcagc 360

<210> 22

<211> 324

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable district of anti-CD28 scFv

<400> 22

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc acgccagcca gaacatctac gtgtggctga actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacaag gccagcaacc tgcacaccgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag ggccagacct acccctacac cttcggcggc 300

ggcaccaagg tggagatcaa gcgc 324

<210> 23

<211> 5

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of junction fragment 1 (Linker 1) in the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 23

Gly Gly Gly Gly Ser

1 5

<210> 24

<211> 15

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of junction fragment 1 (Linker 1) in the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 24

ggtggcggag ggtcc 15

<210> 25

<211> 15

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of junction fragment 2 (Linker 2) in the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 25

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 26

<211> 45

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of junction fragment 2 (Linker 2) in the CD19-CD3-CD28 TsAb_M of monomeric form

<400> 26

ggaggcggag gttccggcgg tgggggatcg gggggtggag ggagt 45

<210> 27

<211> 5

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of junction fragment 1 (Linker 1) in the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 27

Gly Gly Gly Gly Ser

1 5

<210> 28

<211> 15

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of junction fragment 1 (Linker 1) in the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 28

ggtggcggag ggtcc 15

<210> 29

<211> 81

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of junction fragment 2 (Linker 2) in the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 29

Ala Ser Lys Ser Lys Lys Glu Ile Phe Arg Trp Pro Glu Ser Pro Lys

1 5 10 15

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

20 25 30

Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg

35 40 45

Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu

50 55 60

Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly

65 70 75 80

Val

<210> 30

<211> 243

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of junction fragment 2 (Linker 2) in the CD19-CD3-CD28 TsAb_D of dimeric forms

<400> 30

gccagcaaga gcaagaagga gatcttccgc tggcccgaga gccccaaggc ccaggccagc 60

agcgtgccca ccgcccagcc ccaggccgag ggcagcctgg ccaaggccac caccgccccc 120

gccaccaccc gcaacaccgg ccgcggcggc gaggagaaga agaaggagaa ggagaaggag 180

gagcaggagg agcgcgagac caagaccccc gagtgcccca gccacaccca gcccctgggc 240

gtg 243

<210> 31

<211> 19

<212> PRT

<213> Artificial

<220>

<223>The aminoacid sequence of secreting, expressing signal peptide

<400> 31

Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser

1 5 10 15

Ser Arg Ala

<210> 32

<211> 57

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of secreting, expressing signal peptide

<400> 32

atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57

<210> 33

<211> 59

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-Sig-F

<400> 33

gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59

<210> 34

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Sig-R

<400> 34

ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49

<210> 35

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Sig-CD19-F

<400> 35

ctgctggcct cctccagggc cgacatccag ctgacccaga gc 42

<210> 36

<211> 23

<212> DNA

<213> Artificial

<220>

<223> CD19-R

<400> 36

gctgctcacg gtcacggtgg tgc 23

<210> 37

<211> 56

<212> DNA

<213> Artificial

<220>

<223> CD19-G4S-CD3-F

<400> 37

ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag cagagc 56

<210> 38

<211> 20

<212> DNA

<213> Artificial

<220>

<223> CD3-R

<400> 38

cttcagctcc agcttggtgc 20

<210> 39

<211> 86

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD28-F

<400> 39

gcaccaagct ggagctgaag ggaggcggag gttccggcgg tgggggatcg gggggtggag 60

ggagtcaggt gcagctggtg cagagc 86

<210> 40

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD28-R

<400> 40

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 41

<211> 41

<212> DNA

<213> Artificial

<220>

<223> CD3-IgD-F

<400> 41

gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41

<210> 42

<211> 21

<212> DNA

<213> Artificial

<220>

<223> IgD-R

<400> 42

cacgcccagg ggctgggtgt g 21

<210> 43

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-CD28-F

<400> 43

cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42

Claims (16)

1. a kind of three functional molecular, its structure include being incorporated into CD19 the first functional domain, can with reference to and to activate T thin Second functional domain of cellular surface CD3 molecules and can with reference to and activate the 3rd functional domain of T cell surface C D28 molecule.

2. three functional molecular according to claim 1, it is characterised in that three functional molecular can be identification CD19's Meanwhile, with reference to and activate T cell surface C D3 molecule and CD28 molecules, so as to produce the first signal and needed for T cell activation Binary signal.

3. three functional molecular according to claim 1, it is characterised in that first functional domain is the antibody of anti-CD19, Antibody of second functional domain for AntiCD3 McAb, the 3rd functional domain is the antibody of anti-CD28.

4. three functional molecular according to claim 3, it is characterised in that the antibody is selected from Fab antibody, Fv antibody or list Chain antibody.

5. three functional molecular according to claim 1, it is characterised in that first functional domain and second functional domain Between connected by junction fragment 1, connected by junction fragment 2 between second functional domain and the 3rd functional domain.

6. three functional molecular according to claim 5, it is characterised in that the junction fragment 1 and junction fragment 2 selected from Hinge region fragments of the G4S for the junction fragment or Immunoglobulin IgD of unit.

7. three functional molecular according to claim 6, it is characterised in that the aminoacid of the junction fragment in units of G4S Sequence as SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 it is arbitrary shown in；The hinge region of Immunoglobulin IgD The aminoacid sequence of fragment is as shown in SEQ ID NO.29.

8. three functional molecular according to claim 1, it is characterised in that first functional domain is the single-stranded anti-of anti-CD19 Body, single-chain antibody of second functional domain for AntiCD3 McAb, the 3rd functional domain is the single-chain antibody of anti-CD28, described single-stranded Antibody includes weight chain variable district and light chain variable district.

9. three functional molecular according to claim 8, it is characterised in that the weight chain variable of the single-chain antibody of the anti-CD19 The aminoacid sequence in area is as shown in SEQ ID NO.6；The aminoacid sequence of the light chain variable district of the single-chain antibody of the anti-CD19 As shown in SEQ ID NO.7；The aminoacid sequence of the weight chain variable district of the single-chain antibody of the AntiCD3 McAb such as SEQ ID NO.9 institutes Show；The aminoacid sequence of the light chain variable district of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.10；The anti-CD28's The aminoacid sequence of the weight chain variable district of single-chain antibody is as shown in SEQ ID NO.12；The light chain of the single-chain antibody of the anti-CD28 The aminoacid sequence of variable region is as shown in SEQ ID NO.13.

10. three functional molecular according to claim 9, it is characterised in that the aminoacid of the single-chain antibody of the anti-CD19 Sequence is as shown in SEQ ID NO.5；The aminoacid sequence of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.8；It is described anti- The aminoacid sequence of the single-chain antibody of CD28 is as shown in SEQ ID NO.11.

11. three functional moleculars according to claim 1, it is characterised in that the aminoacid sequence of three functional molecular is such as Shown in SEQ ID NO.1 or SEQ ID NO.3.

A kind of 12. polynucleotide, its coding three functional molecular as described in any one of claim 1～11.

A kind of 13. expression vectors, which contains polynucleotide as claimed in claim 12.

A kind of 14. host cells, which is converted by expression vector as claimed in claim 13.

15. as described in any one of claim 1～11 three functional moleculars preparation method, including：Build containing three functional moleculars Then expression vector containing three functional molecular gene orders is converted into host cell induction table by the expression vector of gene order Reach, separate from expression product and obtain three described functional moleculars.

16. three functional moleculars as described in any one of claim 1～11 are used to prepare the purposes of anti-tumor medicine.