CN108264559A - Three functional moleculars and its application of a kind of combination CD19, CD3 and the positive costimulatory molecules of T cell - Google Patents

Abstract

The invention belongs to biomedicine technical fields, and in particular to three functional moleculars and its application of a kind of combination CD19, CD3 and the positive costimulatory molecules of T cell.Three functional molecular, structure includes that the first functional domain of CD19 can be incorporated into, the second functional domain that can combine and activate CD3 and the third functional domain that can combine and activate the positive costimulatory molecules of T cell.The molecule has apparent advantage in terms of preparation process and practical application：The effect of activating T cell being further improved while imparting T cell is positive cell targeted to CD19, the T cell individually mediated during addition is superior to the fragmentation effect of CD19 positive target cells anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies, better than the CAR T technologies of targeting CD19 in the convenience used.

Description

A kind of three functional moleculars of combination CD19, CD3 and the positive costimulatory molecules of T cell and itsUsing

Technical field

The invention belongs to biomedicine technical fields, and in particular to a kind of combination CD19, CD3 and T cell positive costimulation pointThree functional moleculars of son 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 inNormal B lymphocytes surface, CD19 is also high to be expressed in B cell malignant tumour, therefore anti-CD19 monoclonals full length antibody is openedHair is applied to treatment urgency/chronic lymphocytic leukemia and B cell lymphoma (Wang K et al., ExperimentalHematology＆Oncology, 1:36-42,2012).In view of anti-CD19 monoclonal antibodies can not effectively raise cytotoxic T leaching(Cytotoxic T lymphocyte, CTL, the bis- positive T cells of such CD3/CD8 can specific recognition target cell tables for bar cellThe Antigenic Peptide in face/MHC I MHC molecule complex discharges perforin (Peforin), causes target cell lysis dead after autoactivationDie, 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 bispecific antibody (Bi-specific that can be connected T cell and lymthoma B cellAntibody, BsAb) and genetic engineering Chimeric antigen receptor T cell immunotherapy (Chimeric antigen receptorT-cell immunotherapy, CAR-T) (Zhukovsky EA et al., Current Opinion in Immunology,40:24-35,2016).

The bispecific antibody type of the targeting CD19 of current comparative maturity a kind of is the bispecific T of anti-CD19/ AntiCD3 McAbsCell adapter (Bi-specific T cell engager, BiTE), structure is two single-chain antibody (Single-chainVariable fragment, scFv) functional domain is by having flexible connection peptide fragment (Linker) covalently to connect(Goebeler ME et al., Leukemia＆Lymphoma, 57：1021-1032,2016).In the cellular immune processes of body,The TCR/CD3 compounds on CD8 positive T cells surface and antigen presenting cell (Antigen presenting cell, APC) tableSpecific recognition occurs for the endogenous antigen peptide in face/MHC I MHC molecule complex, leads to the cytoplasm section phase of CD3 and co-receptor CD8Interaction activates the protein tyrosine kinase being connected with cytoplasm segment trailer, swashs CD3 cytoplasmic region immunity receptor tyrosine kinaseTyrosine 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 bis- specialHeterogenetic antibody can be formed due to the combination activity with two kinds of antigens of the mankind CD3 and CD19 between T cell and neoplastic B cellCell is connected, while gives T cell primary activation signal, improves its killing targeting to tumour cell.But BiTE is bis-Specific antibody does not have the Fc segments of full length antibody, and molecular weight of albumen is smaller (~54kDa), therefore in the process of oncotherapyIn can pass through hematuria barrier and brain blood barrier, bioavilability is low, and persistent intravenous injection is needed to be administered, while has certain nerveToxicity.

In addition, the activation of T cell needs to rely on dual signal pipeline (Baxter AG et al., Nature in human bodyReviews Immunology, 2：439-446,2002).First, the Antigenic Peptide of APC cell surfaces-MHC molecule compound and TThe TCR/CD3 compounds interaction of cell surface generates the first signal so that T cell primary activation, later APC cell surfacesCostimulatory molecules ligand (such as CD80, CD86,4-1BBL, B7RP-1, OX40L, GITRL, CD40, CD70, PD-L1 and PD-L2 etc.) corresponding with T cell surface costimulatory molecules (Co-stimulatory molecule, for example, CD28,4-1BB, ICOS,OX40, GITR, CD40L, CD27, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT and BTLA etc.) the second letter of interaction generationNumber (costimulatory signal)：Wherein CD28,4-1BB, ICOS, OX40, GITR, CD40L and CD27 etc. belong to positive costimulatory molecules,The second signal (positive costimulatory signal) of generation can lead to the complete activation of T cell；And CTLA-4, PD-1, LAG-3, TIM-3,TIGIT and BTLA etc. belongs to negative costimulatory molecules, mainly lower activation (the negative costimulation letter for terminating T cell that reconciles of effectNumber).Some researches show that only the first signaling pathways can not abundant activating T cell, can cause on the contrary its disability even produceThe T cell of raw activation-inducing is dead (Activation induced cell death, AICD).To solve this problem, it can incite somebody to actionThe bispecific of antitumor antigens/anti-T cell just bispecific antibody of (negative) costimulatory molecules and antitumor antigens/AntiCD3 McAbAntibody combined use, with improve the activation of T cell 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 this method is in actual mechanical processIn there are inconvenience, such as can increase recombination bispecific antibody expression and purification workload and production cost, activation expandThe relative scale of two kinds of bispecific antibodies of optimization is also needed when increasing T cell.In contrast, it is thin preferably to solve T for CAR-T technologiesThe activation problem of born of the same parents.The structure of CAR generally includes：Tumor associated antigen combined area (such as CD19 antigen binding domains, usual sourceIn the scFv segments of anti-CD19 monoclonals full length antibody), extracellular hinge area, transmembrane region and intracellular signal area.Wherein intracellular is believedNumber area is responsible for the activation of mediate T cell, on the one hand completes the first stimulus signal by the Tyrosine Activating Motifs on CD3 ζ chains,On the one hand the expansion of the first stimulus signal is realized by the positive costimulatory signals of CD28, promotes T cell proliferation and activation, and is caused thinIntracellular cytokine secretion increases, Anti-apoptotic proteins secretion increases, cell death postpones etc..But CAR-T technologies in itself there is alsoSome shortcomings：First, the technology dependovirus transfection to T cell carry out genetic modification, complex steps, to experiment condition requirement compared withIt is high；Secondly, the CAR-T cells after amplification in vitro need to be activated when specifically used are fed back in patient body, and dosage control is relatively anti-Body drug has bigger difficulty；It sharply increases in addition, CAR-T cells enter quantity after patient's body and can lead to cytokine storm(Cytokine storm) generates the cell factor of excess in short-term, so as to cause the even death etc. of high fever, low pressure, shockSide reaction.

Invention content

In order to overcome the problems of in the prior art, the purpose of the present invention is to provide one kind in combination with CD19,Three functional moleculars and its application of CD3 and the positive costimulatory molecules of T cell.

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

The first aspect of the present invention, provides a kind of three functional moleculars, and structure includes that the first of CD19 can be incorporated intoFunctional domain, the second functional domain that can be combined and activate T cell surface C D3 molecules and it can combine and T cell is activated just to pierce altogetherSwash the third functional domain of molecule.

Preferably, three functional molecular can while CD19 is identified, with reference to and activate T cell surface C D3 moleculesWith the positive costimulatory molecules of T cell, so as to generate the first signal and the second 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, describedThird functional domain is the antibody of the positive costimulatory molecules of anti-T cell.

Preferably, the antibody is small molecular antibody.

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

Preferably, it is connected between first functional domain and second functional domain by junction fragment 1, second work(It can be connected between domain and the third functional domain by junction fragment 2.

Preferably, the junction fragment 1 and junction fragment 2 are selected from junction fragment or immunoglobulin as unit of G4SThe hinge area segment of IgD.

The G4S is specially GGGGS.The junction fragment as unit of G4S includes one or more G4S units.ExampleSuch as, it is one, two, three or more than four G4S units that can include.In some embodiments of the present invention, one is listedIn the bifunctional molecule of monomeric form, connected between the first functional domain and the second functional domain by the junction fragment 1 as unit of G4SIt connects, is connected between the second functional domain and third functional domain by the junction fragment 2 as unit of G4S.The junction fragment 1 containsOne G4S unit, the amino acid sequence of the junction fragment is as shown in SEQ ID NO.1.There are three the junction fragment 2 containsG4S units, the amino acid sequence of the junction fragment is as shown in SEQ ID NO.3.

The hinge area segment of the Immunoglobulin IgD can be the hinge Ala90-Val170 of Immunoglobulin IgD.ThisIn some embodiments of invention, in the bifunctional molecule for listing a dimeric forms, the first functional domain and the second functional domain itBetween connected by the junction fragment 1 as unit of G4S, pass through Immunoglobulin IgD between the second functional domain and third functional domainThe connection of hinge area segment, the hinge area segment of the Immunoglobulin IgD is the hinge Ala90- of Immunoglobulin IgDVal170.The junction fragment 1 is containing there are one G4S units, the amino acid sequence such as SEQ ID NO.5 institutes of the junction fragmentShow.The amino acid sequence of the junction fragment 2 is as shown in SEQ ID NO.7.The junction fragment 2 can mutually be interconnected by disulfide bondIt connects to form dimer.

Preferably, the C-terminal of first functional domain is connect with the N-terminal of second functional domain；Second functionThe C-terminal in domain is connect with the N-terminal of the third 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 McAbBody, the third functional domain be the positive costimulatory molecules of anti-T cell single-chain antibody, the single-chain antibody include heavy chain variable region andLight chain variable region.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD19 such as SEQ ID NO.41 institutesShow.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.42.The AntiCD3 McAbThe amino acid sequence of the heavy chain variable region of single-chain antibody is as shown in SEQ ID NO.44.The light chain of the single-chain antibody of the AntiCD3 McAbThe amino acid sequence of variable region is as shown in SEQ ID NO.45.

Preferably, the single-chain antibody of the positive costimulatory molecules of anti-T cell can be the single-chain antibody of anti-4-1BB, resistThe single-chain antibody of ICOS, the single-chain antibody of anti-OX40, the single-chain antibody of anti-GITR, the single-chain antibody of anti-CD 40 L or anti-CD27Single-chain antibody it is any.

Preferably, the amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-4-1BB such as SEQ ID NO.47 institutesShow.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-4-1BB is as shown in SEQ ID NO.48.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-ICOS is as shown in SEQ ID NO.50.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of ICOS is as shown in SEQ ID NO.51.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-OX40 is as shown in SEQ ID NO.53.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of OX40 is as shown in SEQ ID NO.54.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-GITR is as shown in SEQ ID NO.56.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of GITR is as shown in SEQ ID NO.57.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD 40 L is as shown in SEQ ID NO.59.It is describedThe amino acid sequence of the light chain variable region of the single-chain antibody of anti-CD 40 L is as shown in SEQ ID NO.60.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD27 is as shown in SEQ ID NO.62.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of CD27 is as shown in SEQ ID NO.63.

In some embodiments of the invention, the amino acid sequence such as SEQ ID of the single-chain antibody of the anti-CD19 are listedShown in NO.40.The amino acid sequence of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.43.The anti-4-1BB's is single-strandedThe amino acid sequence of antibody is as shown in SEQ ID NO.46.The amino acid sequence of the single-chain antibody of the anti-ICOS such as SEQ IDShown in NO.49.The amino acid sequence of the single-chain antibody of the anti-OX40 is as shown in SEQ ID NO.52.The anti-GITR's is single-strandedThe amino acid sequence of antibody is as shown in SEQ ID NO.55.The amino acid sequence of the single-chain antibody of the anti-CD 40 L such as SEQ IDShown in NO.58.The amino acid sequence of the single-chain antibody of the anti-CD27 is as shown in SEQ ID NO.61.

In the preferable case of this case, amino acid sequence such as SEQ ID NO.16, the SEQ of three functional moleculars of monomeric formID NO.20, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO.36 it is any shown in.

In the preferable case of this case, amino acid sequence such as SEQ ID NO.18 of three functional moleculars of dimeric forms,SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.30, SEQ ID NO.34 or SEQ ID NO.38 it is any shown in.

The second aspect of the present invention provides a kind of polynucleotides, encodes aforementioned three functional molecular.

The third aspect of the present invention provides a kind of expression vector, contains foregoing polynucleotides.

The fourth aspect of the present invention provides a kind of host cell, is converted by foregoing expression vectors.

The fifth aspect of the present invention provides a kind of method for preparing aforementioned three functional molecular, including：Structure contains three functionsThen expression vector containing three functional molecular gene orders is converted and is lured into host cell by the expression vector of molecular gene sequenceExpression is led, is detached from expression product and obtains three functional moleculars.

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

The sixth aspect of the present invention provides the purposes that aforementioned three functional molecular is used to prepare anti-tumor medicine.

The seventh aspect of the present invention provides a kind of cancer therapeutics compositions, containing aforementioned three functional molecular and at leastA kind of pharmaceutically acceptable carrier or excipient.The tumour is the tumour that cell surface is the CD19 positives.

The eighth aspect of the present invention discloses a kind of method of external treatment tumour, including by aforementioned three functional molecular orCancer therapeutics compositions are applied to tumor patient.The method can be non-treatment purpose.The tumour is cell tableFace is the tumour of the CD19 positives.

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

(1) three functional molecular of the present invention will be incorporated into the first functional domain of CD19, can combine and activate TSecond functional domain of cell surface CD3 molecules and it can combine and the third functional domain of the positive costimulatory molecules of T cell is activated to mergeIn same protein peptide chain, using eukaryotic cell expression system production, expression product structure is single, and purifying process is easy, albumen productionAmount is high, and preparation process and product are stablized, easy to use；And anti-CD19/ AntiCD3 McAbs bispecific antibody and the anti-T cells of anti-CD19/ are justIf costimulatory molecules bispecific antibody is used in combination, two bispecific antibodies need to distinguish expression and purification, and preparation process is moreComplexity, workload and production cost dramatically increase, and need to optimize the relative scale of the two during use.

(2) three functional molecular of the present invention can generate second (just) stimulus signal of T cell activation, assign TActivation effect to T cell is further improved while cell targeted so that cell factor and Anti-apoptotic proteins pointThe phenomenon that secreting increase, effectively preventing T cell disability and death, the T cell mediated is to the killing energy of CD19 positive target cellsEnough achieve the effect that even better than anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies, and albumen dosage is less.

(3) three functional molecular of the present invention is not related to virus-mediated turning base compared with targeting the CAR-T technologies of CD19Because of operating procedures such as, ex vivo T cell culture and feedbacks, using more convenient, dosage is controllable, into patient's body after cause cell becauseThe risk that son excessively discharges is small, toxic side effect when avoiding using CAR-T.

Description of the drawings

Fig. 1：A. the structure chart of the anti-CD19/ AntiCD3 McAbs of monomeric form/positive costimulatory molecules three-specific antibody of anti-T cell；B.The structure chart of the anti-CD19/ AntiCD3 McAbs of dimeric forms/positive costimulatory molecules three-specific antibody of anti-T cell.

Fig. 2：A. the CD19-CD3-4-1BB TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-4-1BB TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-4-1BB TsAb_M；B. the CD19-CD3-4-1BB TsAb_D SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：AlsoOriginality CD19-CD3-4-1BB TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-4-1BB TsAb_D.

Fig. 3 A：The ELISA qualification results of CD19-CD3-4-1BB TsAb_M, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen 4-1BB-hFc；It is not coated with the measurement result of any antigen.

Fig. 3 B：The ELISA qualification results of CD19-CD3-4-1BB TsAb_D, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen 4-1BB-hFc；It is not coated with the measurement result of any antigen.

Fig. 4：Tri- specific antibodies of CD19-CD3-4-1BB mediation cell killing experiment, using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of CIK (the Cytokine induced killer) cells as the CD3 positives, pointIt Jian Ce not various concentration CD19-CD3-4-1BB TsAb_M, CD19-CD3-4-1BB TsAb_D and CD19-CD3 BsAb institutesThe CIK cell of mediation is to the killing-efficiency of Raji cells；Effector cell：Target cell (E:T ratios)=1：1, kill the time：3h.

Fig. 5：A. the CD19-CD3-ICOS TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-ICOS TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-ICOS TsAb_M；B.The CD19-CD3-ICOS TsAb_D SDS-PAGE analysis charts of purifying, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：ReproducibilityCD19-CD3-ICOS TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-ICOS TsAb_D.

Fig. 6 A：The ELISA qualification results of CD19-CD3-ICOS TsAb_M, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen I COS-hFc；It is not coated with the measurement result of any antigen.

Fig. 6 B：The ELISA qualification results of CD19-CD3-ICOS TsAb_D, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen I COS-hFc；It is not coated with the measurement result of any antigen.

Fig. 7：Tri- specific antibodies of CD19-CD3-ICOS mediation cell killing experiment, using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of the CIK cell as the CD3 positives detect various concentration CD19-CD3- respectivelyThe CIK cell that ICOS TsAb_M, CD19-CD3-ICOS TsAb_D and CD19-CD3 BsAb are mediated is to Raji cellsKilling-efficiency；Effector cell：Target cell (E:T ratios)=1：1, kill the time：3h.

Fig. 8：A. the CD19-CD3-OX40 TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-OX40 TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-OX40 TsAb_M；B.The CD19-CD3-OX40 TsAb_D SDS-PAGE analysis charts of purifying, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：It is non-reducedProperty CD19-CD3-OX40 TsAb_D；Swimming lane 3：Reproducibility CD19-CD3-OX40 TsAb_D.

Fig. 9 A：The ELISA qualification results of CD19-CD3-OX40 TsAb_M, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen OX40-hFc；It is not coated with the measurement result of any antigen.

Fig. 9 B：The ELISA qualification results of CD19-CD3-OX40 TsAb_D, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen OX40-hFc；It is not coated with the measurement result of any antigen.

Figure 10：The cell killing experiment of tri- specific antibodies of CD19-CD3-OX40 mediation.Using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of the CIK cell as the CD3 positives detect various concentration CD19-CD3- respectivelyThe CIK cell that OX40 TsAb_M, CD19-CD3-OX40 TsAb_D and CD19-CD3 BsAb are mediated is to Raji cellsKilling-efficiency；Effector cell：Target cell (E:T ratios)=1：1, kill the time：3h.

Figure 11：A. the CD19-CD3-GITR TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-GITR TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-GITR TsAb_M；B.The CD19-CD3-GITR TsAb_D SDS-PAGE analysis charts of purifying, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：ReproducibilityCD19-CD3-GITR TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-GITR TsAb_D.

Figure 12 A：The ELISA qualification results of CD19-CD3-GITR TsAb_M, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen GITR-hFc；It is not coated with the measurement result of any antigen.

Figure 12 B：The ELISA qualification results of CD19-CD3-GITR TsAb_D, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigen GITR-hFc；It is not coated with the measurement result of any antigen.

Figure 13：Tri- specific antibodies of CD19-CD3-GITR mediation cell killing experiment, using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of the CIK cell as the CD3 positives detect various concentration CD19-CD3- respectivelyThe CIK cell that GITR TsAb_M, CD19-CD3-GITR TsAb_D and CD19-CD3 BsAb are mediated is to Raji cellsKilling-efficiency；Effector cell：Target cell (E:T ratios)=1：1, kill the time：3h.

Figure 14：A. the CD19-CD3-CD40L TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-CD40L TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-CD40L TsAb_M；B. the CD19-CD3-CD40L TsAb_D SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：AlsoOriginality CD19-CD3-CD40L TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-CD40L TsAb_D.

Figure 15 A：The ELISA qualification results of CD19-CD3-CD40L TsAb_M, the curve in figure represent 4 kinds of detections respectivelyAs a result：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；▲ coating 1 μ g/ml weightsGroup antigen CD4 0L-hFc；It is not coated with the measurement result of any antigen.

Figure 15 B：The ELISA qualification results of CD19-CD3-CD40L TsAb_D, the curve in figure represent 4 kinds of detections respectivelyAs a result：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；▲ coating 1 μ g/ml weightsGroup antigen CD4 0L-hFc；It is not coated with the measurement result of any antigen.

Figure 16：Tri- specific antibodies of CD19-CD3-CD40L mediation cell killing experiment, using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of the CIK cell as the CD3 positives detect various concentration CD19-CD3- respectivelyThe CIK cell that CD40L TsAb_M, CD19-CD3-CD40L TsAb_D and CD19-CD3 BsAb are mediated is to Raji cellsKilling-efficiency；Effector cell：Target cell (E:T ratios)=1：1, kill the time：3h.

Figure 17：A. the CD19-CD3-CD27 TsAb_M SDS-PAGE analysis charts purified, swimming lane 1：Molecular weight proteinMarker；Swimming lane 2：Reproducibility CD19-CD3-CD27 TsAb_M；Swimming lane 3：Irreducibility CD19-CD3-CD27 TsAb_M；B.The CD19-CD3-CD27 TsAb_D SDS-PAGE analysis charts of purifying, swimming lane 1：Molecular weight protein Marker；Swimming lane 2：ReproducibilityCD19-CD3-CD27 TsAb_D；Swimming lane 3：Irreducibility CD19-CD3-CD27 TsAb_D.

Figure 18 A：The ELISA qualification results of CD19-CD3-CD27 TsAb_M, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigens c D27-hFc；It is not coated with the measurement result of any antigen.

Figure 18 B：The ELISA qualification results of CD19-CD3-CD27 TsAb_D, the curve in figure represent 4 kinds of detection knots respectivelyFruit：■ is coated with 1 μ g/ml recombinant antigens CD19-hFc；It is coated with 1 μ g/ml recombinant antigens CD3-hFc；The 1 μ g/ml recombinations of ▲ coatingAntigens c D27-hFc；It is not coated with the measurement result of any antigen.

Figure 19：Tri- specific antibodies of CD19-CD3-CD27 mediation cell killing experiment, using Raji lymphoma cells asThe target cell of the CD19 positives, lethal effect cell of the CIK cell as the CD3 positives detect various concentration CD19-CD3- respectivelyThe CIK cell that CD27 TsAb_M, CD19-CD3-CD27 TsAb_D and CD19-CD3 BsAb are mediated is to Raji cellsKilling-efficiency；Effector cell：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：Bispecific 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：Antigen-binding fragment (Fragment of antigen binding)

Fv：Variable region fragment (Variable fragment)

scFv：Single chain variable fragment (Single-chain variable fragment), also known as single-chain antibody

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

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

Linker1：Junction fragment 1

Linker2：Junction fragment 2

Extracellular domain：Extracellular region

CD19-CD3-4-1BB TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti- 4-1BB three-specific antibodies of monomeric form

CD19-CD3-4-1BB TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti- 4-1BB three-specific antibodies of dimeric forms

CD19-CD3-ICOS TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti- ICOS three-specific antibodies of monomeric form

CD19-CD3-ICOS TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti- ICOS three-specific antibodies of dimeric forms

CD19-CD3-OX40 TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti- OX40 three-specific antibodies of monomeric form

CD19-CD3-OX40 TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti- OX40 three-specific antibodies of dimeric forms

CD19-CD3-GITR TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti- GITR three-specific antibodies of monomeric form

CD19-CD3-GITR TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti- GITR three-specific antibodies of dimeric forms

CD19-CD3-CD40L TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti-CD 40 L three-specific antibody of monomeric form

CD19-CD3-CD40L TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti-CD 40 L three-specific antibody of dimeric forms

CD19-CD3-CD27 TsAb_M：Anti- CD19/ AntiCD3 McAbs/anti- CD27 three-specific antibodies of monomeric form

CD19-CD3-CD27 TsAb_D：Anti- CD19/ AntiCD3 McAbs/anti- CD27 three-specific antibodies of dimeric forms

2nd, three functional molecular

Three functional molecular of the present invention, structure include that the first functional domain, the Neng Goujie of CD19 can be incorporated intoMerge the second functional domain of activation T cell surface C D3 molecules and can combine and activate the third work(of the positive costimulatory molecules of T cellIt can domain.

Further, three functional molecular can while CD19 is identified, with reference to and activate T cell surface C D3 pointsSon and the positive costimulatory molecules of T cell, so as to generate the first signal and the second signal needed for T cell activation.

The positive costimulatory molecules of T cell include but not limited to the mankind CD28,4-1BB, ICOS, OX40, GITR, CD40LOr CD27.

The present invention the first functional domain, the second functional domain and third functional domain are had no it is specifically limited, as long as can knowWhile other CD19, with reference to and activate T cell surface C D3 molecules and the positive costimulatory molecules of T cell, so as to generate T cell activationRequired the first signal and the second signal.For example, first functional domain can be the antibody of anti-CD19, second work(Energy domain can be the antibody of AntiCD3 McAb, and the third functional domain can be the antibody of the positive costimulatory molecules of anti-T cell.The antibodyIt can be arbitrary form.But the either antibody of which kind of form, antigen-binding site can containing heavy chain variable region and light chainBecome area.The antibody preferably can be small molecular antibody.The small molecular antibody is the smaller antibody fragment of molecular weight, is resistedFormer binding site includes heavy chain variable region and light chain variable region.Though the molecular weight of the small molecular antibody is small but maintains parent's listAnti- affinity, the specificity for having parent's monoclonal antibody the same.The type of the small molecular antibody mainly includes Fab antibody, Fv resistsBody, single-chain antibody (scFv) etc..Fab antibody is by complete light chain (variable region V_LWith constant region C_L) and Fd sections of (variable region V of heavy chain_HWith the first constant region C_H1) it connects to be formed by disulfide bond.Fv antibody is only connected by the variable region of light chain and heavy chain by non-covalent bondIt connects, is the minimum function fragment that antibody molecule retains intact antigen binding site.Single-chain antibody (scFv) be heavy chain variable region andThe single protein peptide chain molecule that light chain variable region is formed by connecting by junction fragment.

Connected between first functional domain and second functional domain by junction fragment 1, second functional domain andIt is connected between the third functional domain by junction fragment 2.The present invention does not have particular/special requirement for the order of connection, as long as not limitingThe purpose of the present invention.For example, it may be the C-terminal of first functional domain is connect with the N-terminal of second functional domain；The C-terminal of second functional domain is connect with the N-terminal of the third functional domain.The present invention is for junction fragment 1 and connection sheetSection 2 is also without special limitation, as long as do not limit the purpose of the present invention.

Further, the junction fragment 1 and junction fragment 2 are selected from junction fragment or immune globulin as unit of G4SThe hinge area segment of white IgD.

The G4S is specially GGGGS.The junction fragment as unit of G4S includes one or more G4S units.ExampleSuch as, it is one, two, three or more than four G4S units that can include.In some embodiments of the present invention, one is listedIn the bifunctional molecule of monomeric form, connected between the first functional domain and the second functional domain by the junction fragment 1 as unit of G4SIt connects, is connected between the second functional domain and third functional domain by the junction fragment 2 as unit of G4S.The junction fragment 1 containsOne G4S unit, the amino acid sequence of the junction fragment is as shown in SEQ ID NO.1.There are three the junction fragment 2 containsG4S units, the amino acid sequence of the junction fragment is as shown in SEQ ID NO.3.

The hinge area segment of the Immunoglobulin IgD can be the hinge Ala90-Val170 of Immunoglobulin IgD.ThisIn some embodiments of invention, in the bifunctional molecule for listing a dimeric forms, the first functional domain and the second functional domain itBetween connected by the junction fragment 1 as unit of G4S, pass through Immunoglobulin IgD between the second functional domain and third functional domainThe connection of hinge area segment, the hinge area segment of the Immunoglobulin IgD is the hinge Ala90- of Immunoglobulin IgDVal170.The junction fragment 1 is containing there are one G4S units, the amino acid sequence such as SEQ ID NO.5 institutes of the junction fragmentShow.The amino acid sequence of the junction fragment 2 is as shown in SEQ ID NO.7.The junction fragment 2 can mutually be interconnected by disulfide bondIt connects to form dimer.

In the preferred embodiment, the structure diagram of three functional molecular is as shown in Figure 1.Three functionMolecule can be that monomeric form can also be dimeric forms.The structure diagram of three functional moleculars of the monomeric form of the present inventionAs shown in Figure 1A, the first functional domain there are one containing in the structure of three functional molecular with CD19 antigen bindings, one and CD3Second functional domain of antigen binding, one with the third functional domain of the positive costimulatory molecules antigen binding of T cell, first functionDomain is the single-chain antibody (scFv) with CD19 antigen bindings, and second functional domain is the single-chain antibody with CD3 antigen bindings(scFv), the third functional domain is the single-chain antibody (scFv) with the positive costimulatory molecules antigen binding of T cell.The two of the present inventionThe structure diagram of three functional moleculars of dimer form as shown in Figure 1B, in the structure of three functional molecular contain there are two withFirst functional domain of CD19 antigen bindings, two with the second functional domain of CD3 antigen bindings, two with T cell positive costimulation pointThe third functional domain of sub- antigen binding, first functional domain are single-chain antibody (scFv) with CD19 antigen bindings, described theTwo functional domains are the single-chain antibody (scFv) with CD3 antigen bindings, and the third functional domain is resists with the positive costimulatory molecules of T cellThe single-chain antibody (scFv) that original combines.The antigen binding potency of three functional moleculars of the dimeric forms of the present invention is monomeric formTwo times.Due to doubling for the first signal of T cell activation (CD3) and second signal (the positive costimulatory molecules of T cell), cause T thinBorn of the same parents' activation is more abundant, stronger to the fragmentation effect of target cell；CD19 single-chain antibody structural domains double to make it to target cellIdentification is also more accurate, therefore dimer has better using effect compared with monomer.

The positive costimulatory molecules of T cell can be CD28,4-1BB, ICOS, OX40, GITR, CD40L or CD27 etc..

The amino acid sequence of the positive costimulatory molecules mankind CD28 extracellular regions of T cell is as shown in SEQ ID NO.9, specially：

NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP。

The amino acid sequence of the positive costimulatory molecules mankind 4-1BB extracellular regions of T cell is as shown in SEQ ID NO.10, specificallyFor：

LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ。

The amino acid sequence of the positive costimulatory molecules mankind ICOS extracellular regions of T cell is as shown in SEQ ID NO.11, specially：

EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLK。

The amino acid sequence of the positive costimulatory molecules mankind OX40 extracellular regions of T cell is as shown in SEQ ID NO.12, specially：

LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRA。

The amino acid sequence of the positive costimulatory molecules mankind GITR extracellular regions of T cell is as shown in SEQ ID NO.13, specially：

QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEP。

The amino acid sequence of the positive costimulatory molecules mankind CD40L extracellular regions of T cell is as shown in SEQ ID NO.14, specificallyFor：

HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL。

The amino acid sequence of the positive costimulatory molecules mankind CD27 extracellular regions of T cell is as shown in SEQ ID NO.15, specially：

ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR。

Specifically, first functional domain is the single-chain antibody of anti-CD19.The single-chain antibody of the anti-CD19 includes heavy chainVariable region and light chain variable region.The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD19 such as SEQ ID NO.41It is shown.The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.42.Further,The amino acid sequence of the single-chain antibody of the anti-CD19 is as shown in SEQ ID NO.40.

Second functional domain is the single-chain antibody of AntiCD3 McAb.The single-chain antibody of the AntiCD3 McAb is including heavy chain variable region and gentlyChain variable region.The amino acid sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.44.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of CD3 is as shown in SEQ ID NO.45.Further, the AntiCD3 McAbThe amino acid sequence of single-chain antibody is as shown in SEQ ID NO.43.

The third functional domain is the single-chain antibody of the positive costimulatory molecules of anti-T cell.The positive costimulatory molecules of anti-T cellSingle-chain antibody include heavy chain variable region and light chain variable region.

The single-chain antibody of the positive costimulatory molecules of anti-T cell can be the single-chain antibody of anti-4-1BB, anti-ICOS it is single-strandedAntibody, the single-chain antibody of anti-OX40, the single-chain antibody of anti-GITR, the single-chain antibody of anti-CD 40 L or anti-CD27 single-chain antibody itIt is any.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-4-1BB is as shown in SEQ ID NO.47.It is describedThe amino acid sequence of the light chain variable region of the single-chain antibody of anti-4-1BB is as shown in SEQ ID NO.48.The anti-4-1BB's is single-strandedThe amino acid sequence of antibody is as shown in SEQ ID NO.46.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-ICOS is as shown in SEQ ID NO.50.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of ICOS is as shown in SEQ ID NO.51.The single-chain antibody of the anti-ICOSAmino acid sequence as shown in SEQ ID NO.49.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-OX40 is as shown in SEQ ID NO.53.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of OX40 is as shown in SEQ ID NO.54.The single-chain antibody of the anti-OX40Amino acid sequence as shown in SEQ ID NO.52.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-GITR is as shown in SEQ ID NO.56.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of GITR is as shown in SEQ ID NO.57.The single-chain antibody of the anti-GITRAmino acid sequence as shown in SEQ ID NO.55.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD 40 L is as shown in SEQ ID NO.59.It is describedThe amino acid sequence of the light chain variable region of the single-chain antibody of anti-CD 40 L is as shown in SEQ ID NO.60.The anti-CD 40 L it is single-strandedThe amino acid sequence of antibody is as shown in SEQ ID NO.58.

The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD27 is as shown in SEQ ID NO.62.It is described anti-The amino acid sequence of the light chain variable region of the single-chain antibody of CD27 is as shown in SEQ ID NO.63.The single-chain antibody of the anti-CD27Amino acid sequence as shown in SEQ ID NO.61.

In the preferable case of this case, amino acid sequence such as SEQ ID NO.16, the SEQ of three functional moleculars of monomeric formID NO.20, SEQ ID NO.24, SEQ ID NO.28, SEQ ID NO.32 or SEQ ID NO.36 it is any shown in.DimerizationThe amino acid sequence of three functional moleculars of body form such as SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ IDNO.30, SEQ ID NO.34 or SEQ ID NO.38 it is any shown in.But it is not limited to tool cited in preferably case of the inventionBody form.

3rd, the polynucleotides of three functional moleculars are encoded

The polynucleotides of coding three functional molecular of the present invention, can be DNA form or rna form.DNA form packetInclude cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.

The polynucleotides of coding three functional molecular of the present invention, can be by well known to those skilled in the art anyIt is prepared by appropriate technology.The technology sees the general description of this field, such as《Molecular Cloning:A Laboratory guide》(J. Pehanorm BrookersDeng, Science Press, 1995).Including but not limited to recombinant DNA technology, chemical synthesis the methods of；For example, by using overlap-extension PCRPCR methods.

In the preferred embodiment, the nucleotides sequence of the heavy chain variable region of the single-chain antibody of the anti-CD19 is encodedRow are as shown in SEQ ID NO.65.Encode the nucleotide sequence such as SEQ ID of the light chain variable region of the single-chain antibody of the anti-CD19Shown in NO.66.The nucleotide sequence of the single-chain antibody of the anti-CD19 is encoded as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb is encoded as shown in SEQ ID NO.68.It compilesThe nucleotide sequence of the light chain variable region of the single-chain antibody of the code AntiCD3 McAb is as shown in SEQ ID NO.69.Encode the AntiCD3 McAbSingle-chain antibody nucleotide sequence as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-4-1BB is encoded as shown in SEQ ID NO.71.The nucleotide sequence of the light chain variable region of the single-chain antibody of the anti-4-1BB is encoded as shown in SEQ ID NO.72.Described in codingThe nucleotide sequence of the single-chain antibody of anti-4-1BB is as shown in SEQ ID NO.70.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-ICOS is encoded as shown in SEQ ID NO.74.It compilesThe nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-ICOS is as shown in SEQ ID NO.75.It encodes described anti-The nucleotide sequence of the single-chain antibody of ICOS is as shown in SEQ ID NO.73.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-OX40 is encoded as shown in SEQ ID NO.77.It compilesThe nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-OX40 is as shown in SEQ ID NO.78.It encodes described anti-The nucleotide sequence of OX40 single-chain antibodies is as shown in SEQ ID NO.76.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-GITR is encoded as shown in SEQ ID NO.80.It compilesThe nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-GITR is as shown in SEQ ID NO.81.It encodes described anti-The nucleotide sequence of GITR single-chain antibodies is as shown in SEQ ID NO.79.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-CD 40 L is encoded as shown in SEQ ID NO.83.The nucleotide sequence of the light chain variable region of the single-chain antibody of the anti-CD 40 L is encoded as shown in SEQ ID NO.84.Described in codingThe nucleotide sequence of anti-CD 40 L single-chain antibody is as shown in SEQ ID NO.82.

The nucleotide sequence of the heavy chain variable region of the single-chain antibody of the anti-CD27 is encoded as shown in SEQ ID NO.86.It compilesThe nucleotide sequence of the light chain variable region of the single-chain antibody of the code anti-CD27 is as shown in SEQ ID NO.87.It encodes described anti-The nucleotide sequence of CD27 single-chain antibodies is as shown in SEQ ID NO.85.

The nucleotide sequence of junction fragment 1 of the encoding amino acid sequence as shown in SEQ ID NO.1 such as SEQ ID NO.2 institutesShow.

The nucleotide sequence of junction fragment 2 of the encoding amino acid sequence as shown in SEQ ID NO.3 such as SEQ ID NO.4 institutesShow.

The nucleotide sequence of junction fragment 1 of the encoding amino acid sequence as shown in SEQ ID NO.5 such as SEQ ID NO.6 institutesShow.

The nucleotide sequence of junction fragment 2 of the encoding amino acid sequence as shown in SEQ ID NO.7 such as SEQ ID NO.8 institutesShow.

Further, the nucleotide sequence of three functional moleculars of coded cell form such as SEQ ID NO.17, SEQ IDNO.21, SEQ ID NO.25, SEQ ID NO.29, SEQ ID NO.33 or SEQ ID NO.37 it is any shown in.Encode dimerizationThe nucleotide sequence of three functional moleculars of body form such as SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27, SEQ IDNO.31, SEQ ID NO.35 or SEQ ID NO.39 it is any shown in.

4th, expression vector

The expression vector of the present invention contains the polynucleotides for encoding three functional molecular.Those skilled in the artWell known method can be used to build the expression vector.These methods include recombinant DNA technology, DNA synthetic technologys etc..It can will compileThe DNA of the code fusion protein is effectively connected in the multiple cloning sites in carrier, mRNA to be instructed to synthesize and then expresses albumen,Or for homologous recombination.In the preferable case of the present invention, the expression vector uses pcDNA3.1.The host cell usesChinese 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 ordersUp to carrier, the expression vector containing three functional molecular gene orders is then converted into host cell induced expression, is produced from expressionSeparation obtains three functional moleculars in object.In the preferable case of the present invention, the expression vector uses pcDNA3.1.It is describedHost cell uses 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.The tumour is that cell surface is the swollen of the CD19 positivesKnurl.

In present pre-ferred embodiments, be found by experiment that, three functional moleculars of the invention be respectively provided with CD19, CD3 andThe external of the positive costimulatory molecules recombinant antigen of corresponding T cell combines activity, can promote targeting of the T cell to CD19 positive target cellsKilling, and dimer has better effect compared with monomer.

7th, cancer therapeutics compositions

The cancer therapeutics compositions of the present invention, it is pharmaceutically acceptable containing aforementioned three functional molecular and at least oneCarrier or excipient.The tumour is the tumour that cell surface is the CD19 positives.

Pharmaceutical composition provided by the present invention can exist with a variety of dosage forms, such as the injection for intravenous injection, useIn the transdermic absorbent of hypodermic injection, epidermis external application etc., for spraying the spray of nose, larynx, oral cavity, epidermis, mucous membrane etc., for drippingThe drops of nose, eye, ear etc., it is a variety of for the suppository of anal intestine etc., tablet, pulvis, granula, capsule, oral liquid, paste, creme etc.The composition of form and pulmonary administration preparation and other parenterai administrations.The drug of above-mentioned various dosage forms can be according to pharmacyIt is prepared by the conventional method in field.

The diluent of the carrier including pharmaceutical field routine, excipient, filler, adhesive, wetting agent, disintegrant,Sorbefacient, surfactant, absorption carrier, lubricant etc..The Pharmaceutical composition can also add in flavouring agent, sweetenerDeng.

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

8th, the method for external treatment tumour

The method of the external treatment tumour of the present invention, including aforementioned three functional molecular or cancer therapeutics compositions are appliedFor in tumor patient.The tumour is the tumour that cell surface is the CD19 positives.The method can be non-treatment purpose.It in present pre-ferred embodiments, is found by experiment that, three functional moleculars of the invention are respectively provided with and CD19, CD3 and corresponding T cellThe external of positive costimulatory molecules recombinant antigen combines activity, can promote target killing of the T cell to CD19 positive target cells, andDimer has better effect compared with monomer.

The present invention is directed to anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies and targets the deficiency of the CAR-T technologies of CD19,CD19 can be identified simultaneously by being constructed by the method for genetic engineering and antibody engineering, CD3 and the positive costimulatory molecules of any T cellThree functional moleculars.The molecule has apparent advantage in terms of preparation process and practical application：T cell is being assigned to the CD19 positivesThe effect of activating T cell is further improved while cell targeted, the T cell individually mediated during addition is to the CD19 positivesThe fragmentation effect of target cell is superior to anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies, better than targeting in the convenience usedThe CAR-T technologies of CD19.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to downState specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describeEmbodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example,Usually according to normal condition or the condition proposed by according to each manufacturer.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical rangeAny one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using andScientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,Outside material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use and thisAny method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come realThe existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology leadMolecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology andThe routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；theSeries METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1：The structure of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D carrier for expression of eukaryonIt builds

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules 4-1BB albumen is named as CD19-CD3-4-1BB TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-4-1BB TsAb_M and the design of CD19-CD3-4-1BB TsAb_D constructing plans

The specific constructing plans of CD19-CD3-4-1BB TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-4-1BB scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, then pass through junction fragment 2 between AntiCD3 McAb scFv and anti-4-1BB scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-4-1BB TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAbScFv is connected with the sequence of anti-4-1BB scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAbIt is connected between scFv by junction fragment 1 (Linker 1), with IgD hinges between AntiCD3 McAb scFv and anti-4-1BB scFv sequencesArea (Ala 90-Val 170) is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, anti-4-1BB scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65, specially：

CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66, specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG。

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64, specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC。

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69, specially：

GACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67, specially：

GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG。

The nucleotide sequence of the heavy chain variable region of anti-4-1BB scFv is as shown in SEQ ID NO.71, specially：

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-4-1BB scFv is as shown in SEQ ID NO.72, specially：

GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of anti-4-1BB scFv is as shown in SEQ ID NO.70, specially：

CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence such as SEQ of the CD19-CD3-4-1BB TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in ID NO.2, specially：GGTGGCGGAGGGTCC.

The nucleotide sequence such as SEQ of the CD19-CD3-4-1BB TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in ID NO.4, specially：

GGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGT。

The nucleotide sequence such as SEQ of the CD19-CD3-4-1BB TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6, specially：GGTGGCGGAGGGTCC.

The nucleotide sequence such as SEQ of the CD19-CD3-4-1BB TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8, specially：

GCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTG。

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88, specially：

MTRLTVLALLAGLLASSRA。

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89, specially：

ATGACCCGGCTGACCGTGCTGGCCCTGCTGGCCGGCCTGCTGGCCTCCTCCAGGGCC。

2nd, CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D construction of eukaryotic expression vector

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

It is built for the clone of CD19-CD3-4-1BB TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- 4-1BB-F and pcDNA3.1-4-1BB-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of the anti-4-1BB scFv of Linker 2+；For the clone of CD19-CD3-4-1BB TsAb_DStructure, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, is then utilized respectively primerSig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-4-1BB-F andPcDNA3.1-4-1BB-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker2nd, the gene order of anti-4-1BB scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from WuJiang Jinan protein Science and Technology Ltd.) splicing monomer and dimeric forms three-specific antibody full-length gene order have no respectivelySeam is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process, converts bacillus coli DH 5 alpha, is utilizedBacterium colony PCR carries out positive clone identification, is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.It will then be sequenced justTrue recon (recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-4-1BB TsAb_M of monomeric form and the CD19-CD3-4- of dimeric formsThe full-length gene order of 1BB TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-4-1BB TsAb_M of monomeric form is as shown in SEQ ID NO.17,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of the CD19-CD3-4-1BB TsAb_D of dimeric forms is as shown in SEQ ID NO.19, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCCGCCAGAGCCCCGAGAAGGGCCTGGAGTGGATCGGCGAGATCAACCACGGCGGCTACGTGACCTACAACCCCAGCCTGGAGAGCCGCGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGCGACTACGGCCCCGGCAACTACGACTGGTACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCTGCCGCGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCCGCCTGCTGATCTACGACGCCAGCAACCGCGCCACCGGCATCCCCGCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCGCAGCAACTGGCCCCCCGCCCTGACCTTCTGCGGCGGCACCAAGGTGGAGATCAAGCGC。

The primer used in table 1.CD19-CD3-4-1BB three-specific antibody gene clonings

Embodiment 2：The expression and purification of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D

First, the expression of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BBTsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombinant plasmid in Example 1：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollect culture supernatant and carry out destination protein detection of expression.

2nd, the purifying of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in Figure 2 under reduction and non reducing conditions.It can be seen from the figure that through Protein L affinity chromatographysAfter column purification, the purity of CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D recombinant proteins is equal>95%：ItsThe theoretical molecular weight of middle CD19-CD3-4-1BB TsAb_M recombinant proteins is 80.6kDa, reduction and the albumen under non reducing conditionsSingle electrophoretic band is presented, molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Fig. 2A)；CD19-The theoretical molecular weight of CD3-4-1BB TsAb_D recombinant proteins is 88.4kDa, and the protein electrophoresis band is presented under reducing conditionMolecular weight is consistent with monomer, and molecular weight (about 180kDa) consistent with dimer (figure is presented in electrophoretic band under non reducing conditions2B), illustrate that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibody is twoDimer form.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-4-1BB TsAb_M is monomeric form, and CD19-CD3-4-1BB TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.16 of the CD19-CD3-4-1BB TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

The amino acid sequence of the CD19-CD3-4-1BB TsAb_D of dimeric forms is as shown in SEQ ID NO.18, specificallyFor：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41, specially：

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS。

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK。

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44, specially：

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS。

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45, specially：

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK。

The amino acid sequence of anti-4-1BB scFv is as shown in SEQ ID NO.46, specially：

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

The amino acid sequence of the heavy chain variable region of anti-4-1BB scFv is as shown in SEQ ID NO.47, specially：

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-4-1BB scFv is as shown in SEQ ID NO.48, specially：

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFCGGTKVEIKR。

The amino acid sequence such as SEQ of the CD19-CD3-4-1BB TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in ID NO.1, specially：GGGGS.

The amino acid sequence such as SEQ of the CD19-CD3-4-1BB TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in ID NO.3, specially：GGGGSGGGGSGGGGS.

The amino acid sequence such as SEQ of the CD19-CD3-4-1BB TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5, specially：GGGGS.

The amino acid sequence such as SEQ of the CD19-CD3-4-1BB TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7, specially：

ASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGV。

Embodiment 3：The antigen knot of ELISA detection CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_DClose activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind 4-1BB-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-4-1BB TsAb_M or CD19-CD3-4-1BB purified with 10 μ g/mlTsAb_D carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as shown in Figure 3A and Figure 3B：Fig. 3 A illustrate CD19-CD3-4-1BB TsAb_M and recombinant antigenCD19-hFc, CD3-hFc and 4-1BB-hFc are respectively provided with external combination activity, and wherein 4-1BB combines active highest, and CD19 is combinedActivity is taken second place, and it is weaker that CD3 combines activity；Fig. 3 B illustrate CD19-CD3-4-1BB TsAb_D and recombinant antigen CD19-hFc, CD3-HFc and 4-1BB-hFc equally has external combination activity, and wherein 4-1BB combines active highest, and CD19 combination activity is taken second place, CD3It is weaker with reference to activity.

Embodiment 4：The cell killing experiment of CD19-CD3-4-1BB three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-4-1BB TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-4-1BB TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-4-1BB TsAb_M and CD19-CD3-4-1BB TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of trainings altogetherAfter supporting 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to following public affairsFormula calculates cell killing efficiency, and every group of experiment repeats detection 3 times；Simultaneously using be not added with the cell killing efficiency of any antibody asBlank control.

The results are shown in Figure 4：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any antibodyUnder conditions of, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell significantly increases the killing-efficiency of Raji cells, wherein CD19-CD3-4-The Cell killing efficacy that 1BB TsAb_D are mediated is best, and killing-efficiency respectively may be about 96%, 96% and 92%, CD19-CD3-The effect of 4-1BB TsAb_M is taken second place, and killing-efficiency is about 92%, 90% and the effect of 86%, CD19-CD3BsAb are most weak, is killedHinder efficiency and respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml), CD19-The CIK cell that CD3-4-1BB TsAb_D and CD19-CD3-4-1BB TsAb_M are mediated to the killing-efficiencies of Raji cells stillHave and significantly improve, killing-efficiency respectively may be about 87% and 80%, and CD19-CD3 BsAb do not have substantially compared with blank controlEffect.The above results illustrate that the T cell that tri- specific antibodies of CD19-CD3-4-1BB TiTE of two kinds of forms are mediated is positive to CD19The target killing activity of property tumour cell is superior to CD19-CD3 BiTE bispecific antibodies, and wherein dimeric forms are compared with monomerForm has better effect.

Embodiment 5：The structure of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D carrier for expression of eukaryon

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules ICOS albumen is named as CD19-CD3-ICOS TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-ICOS TsAb_M and the design of CD19-CD3-ICOS TsAb_D constructing plans

The specific constructing plans of CD19-CD3-ICOS TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFv andThe sequence of anti-ICOS scFv is connected by junction fragment (Linker), specifically, between anti-CD19 scFv and AntiCD3 McAb scFvIt is connected by junction fragment 1 (Linker 1), then passes through junction fragment 2 between AntiCD3 McAb scFv and anti-ICOS scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-ICOS TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-ICOS scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, with IgD hinge areas (Ala between AntiCD3 McAb scFv and anti-ICOS scFv sequences90-Val 170) it is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, resistICOS scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65.

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66.

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68.

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69.

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of anti-ICOS scFv is as shown in SEQ ID NO.74, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-ICOS scFv is as shown in SEQ ID NO.75, specially：

GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence of anti-ICOS scFv is as shown in SEQ ID NO.73, specially：

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence such as SEQ ID of the CD19-CD3-ICOS TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.2.

The nucleotide sequence such as SEQ ID of the CD19-CD3-ICOS TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.4.

The nucleotide sequence such as SEQ of the CD19-CD3-ICOS TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6.

The nucleotide sequence such as SEQ of the CD19-CD3-ICOS TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8.

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89.

2nd, CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1(being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the three-specific antibody of dimeric forms, separately designPrimer as shown in table 2, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplificationZhou Hongxun Science and Technology Ltd.s synthesize.

It is built for the clone of CD19-CD3-ICOS TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- ICOS-F and pcDNA3.1-ICOS-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of the anti-ICOS scFv of Linker 2+；For clone's structure of CD19-CD3-ICOS TsAb_DIt builds, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-ICOS-F and pcDNA3.1-ICOS-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker 2, anti-ICOSThe gene order of scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from Wujiang offshore albumenMatter Science and Technology Ltd.) splice respectively monomer and dimeric forms three-specific antibody full-length gene order and it is seamless be cloned into throughOn the pcDNA3.1 expression vectors of EcoRI and HindIII linearization process, bacillus coli DH 5 alpha is converted, is carried out using bacterium colony PCRPositive clone identification is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.Correct recon will then be sequenced(recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-ICOS TsAb_M of monomeric form and the CD19-CD3-ICOS of dimeric formsThe full-length gene order of TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-ICOS TsAb_M of monomeric form is as shown in SEQ ID NO.21,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence of the CD19-CD3-ICOS TsAb_D of dimeric forms is as shown in SEQ ID NO.23, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGCGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCAACCCCCACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGGCCGCGTGACCATGACCCGCGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCCGCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCACCTACTACTACGACAGCAGCGGCTACTACCACGACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCCTGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCAACAGCTTCCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The primer used in table 2.CD19-CD3-ICOS three-specific antibody gene clonings

Embodiment 6：The expression and purification of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D

First, the expression of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombinant plasmid in Example 5：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollect culture supernatant and carry out destination protein detection of expression.

2nd, the purifying of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in Figure 5 under reduction and non reducing conditions.It can be seen from the figure that through Protein L affinity chromatographysAfter column purification, the purity of CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D recombinant proteins is equal>95%：WhereinThe theoretical molecular weight of CD19-CD3-ICOS TsAb_M recombinant proteins is 80.7kDa, and the albumen is equal under reduction and non reducing conditionsSingle electrophoretic band is presented, molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Fig. 5 A)；CD19-The theoretical molecular weight of CD3-ICOS TsAb_D recombinant proteins is 88.6kDa, and the protein electrophoresis band is presented point under reducing conditionSon amount is consistent with monomer, and molecular weight (about 180kDa) consistent with dimer (Fig. 5 B) is presented in electrophoretic band under non reducing conditions,Illustrate that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibody is dimerForm.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-ICOS TsAb_MFor monomeric form, CD19-CD3-ICOS TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.20 of the CD19-CD3-ICOS TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

The amino acid sequence of the CD19-CD3-ICOS TsAb_D of dimeric forms is as shown in SEQ ID NO.22, specificallyFor：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40.

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41.

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42.

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45.

The amino acid sequence of anti-ICOS scFv is as shown in SEQ ID NO.49, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

The amino acid sequence of the heavy chain variable region of anti-ICOS scFv is as shown in SEQ ID NO.50, specially：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSS。

The amino acid sequence of the light chain variable region of anti-ICOS scFv is as shown in SEQ ID NO.51, specially：

DIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK。

The amino acid sequence such as SEQ ID of the CD19-CD3-ICOS TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.1.

The amino acid sequence such as SEQ ID of the CD19-CD3-ICOS TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.3.

The amino acid sequence such as SEQ of the CD19-CD3-ICOS TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5.

The amino acid sequence such as SEQ of the CD19-CD3-ICOS TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7.

Embodiment 7：The antigen binding of ELISA detection CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_DActivity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind ICOS-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-ICOS TsAb_M or CD19-CD3-ICOS purified with 10 μ g/mlTsAb_D carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as shown in Figure 6 A and 6 B：Fig. 6 A illustrate CD19-CD3-ICOS TsAb_M and recombinant antigen CD19-HFc, CD3-hFc and ICOS-hFc are respectively provided with external combination activity, and wherein ICOS combines active highest, and CD19 is combined active timeIt, it is weaker that CD3 combines activity；Fig. 6 B illustrate CD19-CD3-ICOS TsAb_D and recombinant antigen CD19-hFc, CD3-hFc andICOS-hFc equally has external combination activity, and wherein ICOS combines active highest, and CD19 combination activity is taken second place, and CD3 combines workProperty is weaker.

Embodiment 8：The cell killing experiment of CD19-CD3-ICOS three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-ICOS TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-ICOS TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-ICOS TsAb_M and CD19-CD3-ICOS TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of co-cultivationsAfter 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to the following formulaCell killing efficiency is calculated, every group of experiment repeats detection 3 times；Simultaneously to be not added with the cell killing efficiency of any antibody as emptyWhite control.

The results are shown in Figure 7：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any antibodyUnder conditions of, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell significantly increases the killing-efficiency of Raji cells, wherein CD19-CD3-The Cell killing efficacy that ICOS TsAb_D are mediated is best, and killing-efficiency respectively may be about 94%, 94% and 82%, CD19-CD3-The effect of ICOS TsAb_M is taken second place, and killing-efficiency is about 92%, 86% and the effect of 84%, CD19-CD3 BsAb are most weak, is killedHinder efficiency and respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml), CD19-The CIK cell that CD3-ICOS TsAb_D and CD19-CD3-ICOS TsAb_M are mediated still has the killing-efficiency of Raji cellsIt significantly improves, killing-efficiency respectively may be about 76% and 71%, and CD19-CD3 BsAb are not imitated substantially compared with blank controlFruit.The above results illustrate that the T cell that tri- specific antibodies of CD19-CD3-ICOS TiTE of two kinds of forms are mediated is positive to CD19The target killing activity of tumour cell is superior to CD19-CD3 BiTE bispecific antibodies, and wherein dimeric forms are compared with monomer shapeFormula has better effect.

Embodiment 9：The structure of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D carrier for expression of eukaryon

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules OX40 albumen is named as CD19-CD3-OX40 TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-OX40 TsAb_M and the design of CD19-CD3-OX40 TsAb_D constructing plans

The specific constructing plans of CD19-CD3-OX40 TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFv andThe sequence of anti-OX40 scFv is connected by junction fragment (Linker), specifically, between anti-CD19 scFv and AntiCD3 McAb scFvIt is connected by junction fragment 1 (Linker 1), then passes through junction fragment 2 between AntiCD3 McAb scFv and anti-OX40 scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-OX40 TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-OX40 scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, with IgD hinge areas (Ala between AntiCD3 McAb scFv and anti-OX40 scFv sequences90-Val 170) it is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, resistOX40scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65.

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66.

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68.

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69.

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of anti-OX40 scFv is as shown in SEQ ID NO.77, specially：

CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-OX40 scFv is as shown in SEQ ID NO.78, specially：

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

The nucleotide sequence of anti-OX40 scFv is as shown in SEQ ID NO.76, specially：

CAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

The nucleotide sequence such as SEQ ID of the CD19-CD3-OX40 TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.2.

The nucleotide sequence such as SEQ ID of the CD19-CD3-OX40 TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.4.

The nucleotide sequence such as SEQ of the CD19-CD3-OX40 TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6.

The nucleotide sequence such as SEQ of the CD19-CD3-OX40 TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8.

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89.

2nd, CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1(being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the three-specific antibody of dimeric forms, separately designPrimer as shown in table 3, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplificationZhou Hongxun Science and Technology Ltd.s synthesize.

It is built for the clone of CD19-CD3-OX40 TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- OX40-F and pcDNA3.1-OX40-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of the anti-OX40 scFv of Linker 2+；For clone's structure of CD19-CD3-OX40 TsAb_DIt builds, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-OX40-F and pcDNA3.1-OX40-R amplifies anti-CD19 s cFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker 2, anti-OX40The gene order of scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from Wujiang offshore albumenMatter Science and Technology Ltd.) splice respectively monomer and dimeric forms three-specific antibody full-length gene order and it is seamless be cloned into throughOn the pcDNA3.1 expression vectors of EcoRI and HindIII linearization process, bacillus coli DH 5 alpha is converted, is carried out using bacterium colony PCRPositive clone identification is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.Correct recon will then be sequenced(recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-OX40 TsAb_M of monomeric form and the CD19-CD3- of dimeric formsThe full-length gene order of OX40TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-OX40 TsAb_M of monomeric form is as shown in SEQ ID NO.25,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

The nucleotide sequence of the CD19-CD3-OX40 TsAb_D of dimeric forms is as shown in SEQ ID NO.27, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCAGCAGCAGCAGCAGCACCATCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGACGAGGACACCGCCGTGTACTACTGCGCCCGCGGCGTGTACCACAACGGCTGGAGCTTCTTCGACTACTGGGGCCAGGGCACCCTGCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCAACCGCGTGACCATCACCTGCCGCGCCAGCCAGGACATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGCACCCGCCTGGAGATCAAGCGC。

The primer used in table 3.CD19-CD3-OX40 three-specific antibody gene clonings

Embodiment 10：The expression and purification of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D

First, the expression of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombinant plasmid in Example 9：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollect culture supernatant and carry out destination protein detection of expression.

2nd, the purifying of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in Figure 8 under reduction and non reducing conditions.It can be seen from the figure that through Protein L affinity chromatographysAfter column purification, the purity of CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D recombinant proteins is equal>95%：WhereinThe theoretical molecular weight of CD19-CD3-OX40 TsAb_M recombinant proteins is 80.1kDa, and the albumen is equal under reduction and non reducing conditionsSingle electrophoretic band is presented, molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Fig. 8 A)；CD19-The theoretical molecular weight of CD3-OX40 TsAb_D recombinant proteins is 88.0kDa, and the protein electrophoresis band is presented point under reducing conditionSon amount is consistent with monomer, and molecular weight (about 180kDa) consistent with dimer (Fig. 8 B) is presented in electrophoretic band under non reducing conditions,Illustrate that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibody is dimerForm.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-OX40 TsAb_MFor monomeric form, CD19-CD3-OX40 TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.24 of the CD19-CD3-OX40 TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

The amino acid sequence of the CD19-CD3-OX40 TsAb_D of dimeric forms is as shown in SEQ ID NO.26, specificallyFor：DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40.

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41.

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42.

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45.

The amino acid sequence of anti-OX40 scFv is as shown in SEQ ID NO.52, specially：

QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

The amino acid sequence of the heavy chain variable region of anti-OX40 scFv is as shown in SEQ ID NO.53, specially：

QLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGVYHNGWSFFDYWGQGTLLTVSS。

The amino acid sequence of the light chain variable region of anti-OX40 scFv is as shown in SEQ ID NO.54, specially：

DIQMTQSPSSLSASVGNRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTRLEIKR。

The amino acid sequence such as SEQ ID of the CD19-CD3-OX40 TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.1.

The amino acid sequence such as SEQ ID of the CD19-CD3-OX40 TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.3.

The amino acid sequence such as SEQ of the CD19-CD3-OX40 TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5.

The amino acid sequence such as SEQ of the CD19-CD3-OX40 TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7.

Embodiment 11：The antigen knot of ELISA detection CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_DClose activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind OX40-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-OX40 TsAb_M or CD19-CD3-OX40TsAb_ purified with 10 μ g/mlD carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as shown in fig. 9 a and fig. 9b：Fig. 9 A illustrate CD19-CD3-OX40 TsAb_M and recombinant antigen CD19-HFc, CD3-hFc and OX40-hFc are respectively provided with external combination activity, and wherein CD19 combines active highest, and OX40 is combined active timeIt, it is weaker that CD3 combines activity；Fig. 9 B illustrate CD19-CD3-OX40 TsAb_D and recombinant antigen CD19-hFc, CD3-hFc andOX40-hFc equally has external combination activity, and wherein CD19 combines active highest, and OX40 combination activity is taken second place, and CD3 combines workProperty is weaker.

Embodiment 12：The cell killing experiment of CD19-CD3-OX40 three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-OX40 TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-OX40 TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-OX40 TsAb_M and CD19-CD3-OX40 TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of co-cultivationsAfter 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to the following formulaCell killing efficiency is calculated, every group of experiment repeats detection 3 times；Simultaneously to be not added with the cell killing efficiency of any antibody as emptyWhite control.

The results are shown in Figure 10：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any resistUnder conditions of body, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell significantly increases the killing-efficiency of Raji cells, wherein CD19-CD3-The Cell killing efficacy that OX40 TsAb_D are mediated is best, and killing-efficiency respectively may be about 96%, 92% and 86%, CD19-CD3-The effect of OX40 TsAb_M is taken second place, and killing-efficiency is about 89%, 82% and the effect of 80%, CD19-CD3 BsAb are most weak, is killedHinder efficiency and respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml), CD19-The CIK cell that CD3-OX40 TsAb_D and CD19-CD3-OX40 TsAb_M are mediated still has the killing-efficiency of Raji cellsIt significantly improves, killing-efficiency respectively may be about 72% and 68%, and CD19-CD3 BsAb are not imitated substantially compared with blank controlFruit.The above results illustrate that the T cell that tri- specific antibodies of CD19-CD3-OX40 TiTE of two kinds of forms are mediated is positive to CD19The target killing activity of tumour cell is superior to CD19-CD3 BiTE bispecific antibodies, and wherein dimeric forms are compared with monomer shapeFormula has better effect.

Embodiment 13：The structure of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D carrier for expression of eukaryon

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules GITR albumen is named as CD19-CD3-GITR TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-GITR TsAb_M and the design of CD19-CD3-GITR TsAb_D constructing plans

The specific constructing plans of CD19-CD3-GITR TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFv andThe sequence of anti-GITR scFv is connected by junction fragment (Linker), specifically, between anti-CD19 scFv and AntiCD3 McAb scFvIt is connected by junction fragment 1 (Linker 1), then passes through junction fragment 2 between AntiCD3 McAb scFv and anti-GITR scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-GITR TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-GITR scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, with IgD hinge areas (Ala between AntiCD3 McAb scFv and anti-GITR scFv sequences90-Val 170) it is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, resistGITR scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65.

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66.

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68.

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69.

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of anti-GITR scFv is as shown in SEQ ID NO.80, specially：

CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-GITR scFv is as shown in SEQ ID NO.81, specially：

GACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

The nucleotide sequence of anti-GITR scFv is as shown in SEQ ID NO.79, specially：

CAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

The nucleotide sequence such as SEQ ID of the CD19-CD3-GITR TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.2.

The nucleotide sequence such as SEQ ID of the CD19-CD3-GITR TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.4.

The nucleotide sequence such as SEQ of the CD19-CD3-GITR TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6.

The nucleotide sequence such as SEQ of the CD19-CD3-GITR TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8.

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89.

2nd, CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1(being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the three-specific antibody of dimeric forms, separately designPrimer as shown in table 4, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplificationZhou Hongxun Science and Technology Ltd.s synthesize.

It is built for the clone of CD19-CD3-GITR TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- GITR-F and pcDNA3.1-GITR-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of the anti-GITR scFv of Linker 2+；For clone's structure of CD19-CD3-GITR TsAb_DIt builds, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-GITR-F and pcDNA3.1-GITR-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker 2, anti-GITRThe gene order of scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from Wujiang offshore albumenMatter Science and Technology Ltd.) splice respectively monomer and dimeric forms three-specific antibody full-length gene order and it is seamless be cloned into throughOn the pcDNA3.1 expression vectors of EcoRI and HindIII linearization process, bacillus coli DH 5 alpha is converted, is carried out using bacterium colony PCRPositive clone identification is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.Correct recon will then be sequenced(recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-GITR TsAb_M of monomeric form and the CD19-CD3-GITR of dimeric formsThe full-length gene order of TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-GITR TsAb_M of monomeric form is as shown in SEQ ID NO.29,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

The nucleotide sequence of the CD19-CD3-GITR TsAb_D of dimeric forms is as shown in SEQ ID NO.31, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGACCCTGAAGGAGAGCGGCCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATCCGCCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACAAGTACTACAACCCCAGCCTGAAGAGCCAGCTGACCATCAGCAAGGACACCAGCCGCAACCAGGTGTTCCTGAAGATCACCAGCGTGGACACCGCCGACGCCGCCACCTACTACTGCGCCCGCACCCGCCGCTACTTCCCCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCAGAAGTTCATGAGCACCAGCGTGGGCGACCGCGTGAGCGTGACCTGCAAGGCCAGCCAGAACGTGGGCACCAACGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGGCCCTGATCTACAGCGCCAGCTACCGCTACAGCGGCGTGCCCGACCGCTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAACGTGCACAGCGAGGACCTGGCCGAGTACTTCTGCCAGCAGTACAACACCGACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG。

The primer used in table 4.CD19-CD3-GITR three-specific antibody gene clonings

Embodiment 14：The expression and purification of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D

First, the expression of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombinant plasmid in Example 13：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollection

Culture supernatant carries out destination protein detection of expression

2nd, the purifying of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-GITR TsAb_M and CD19-CD3-GITR the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in figure 11 under reduction and non reducing conditions.It can be seen from the figure that through the affine layers of Protein LAfter analysing column purification, the purity of CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D recombinant proteins is equal>95%：ItsThe theoretical molecular weight of middle CD19-CD3-GITR TsAb_M recombinant proteins is 80.1kDa, reduction and the albumen under non reducing conditionsSingle electrophoretic band is presented, molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Figure 11 A)；CD19-The theoretical molecular weight of CD3-GITR TsAb_D recombinant proteins is 88.0kDa, and the protein electrophoresis band is presented point under reducing conditionSon amount is consistent with monomer, and molecular weight (about 180kDa) consistent with dimer (Figure 11 B) is presented in electrophoretic band under non reducing conditions,Illustrate that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibody is dimerForm.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-GITR TsAb_MFor monomeric form, CD19-CD3-GITR TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.28 of the CD19-CD3-GITR TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

The amino acid sequence of the CD19-CD3-GITR TsAb_D of dimeric forms is as shown in SEQ ID NO.30, specificallyFor：DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40.

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41.

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42.

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45.

The amino acid sequence of anti-GITR scFv is as shown in SEQ ID NO.55, specially：

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

The amino acid sequence of the heavy chain variable region of anti-GITR scFv is as shown in SEQ ID NO.56, specially：

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-GITR scFv is as shown in SEQ ID NO.57, specially：

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK。

The amino acid sequence such as SEQ ID of the CD19-CD3-GITR TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.1.

The amino acid sequence such as SEQ ID of the CD19-CD3-GITR TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.3.

The amino acid sequence such as SEQ of the CD19-CD3-GITR TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5.

The amino acid sequence such as SEQ of the CD19-CD3-GITR TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7.

Embodiment 15：The antigen knot of ELISA detection CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_DClose activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind GITR-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-GITR TsAb_M or CD19-CD3-GITR purified with 10 μ g/mlTsAb_D carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as illustrated in figs. 12 a and 12b：Figure 12 A illustrate CD19-CD3-GITR TsAb_M and recombinant antigenCD19-hFc, CD3-hFc and GITR-hFc are respectively provided with external combination activity, and wherein GITR and CD19 combine higher, the CD3 of activityIt is weaker with reference to activity；Figure 12 B illustrate CD19-CD3-GITR TsAb_D and recombinant antigen CD19-hFc, CD3-hFc and GITR-HFc is similary to have external combination activity, and wherein GITR and CD19 combination activity are higher, and CD3 combines active weaker.

Embodiment 16：The cell killing experiment of CD19-CD3-GITR three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-GITR TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-GITR TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-GITR TsAb_M and CD19-CD3-GITR TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of co-cultivationsAfter 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to the following formulaCell killing efficiency is calculated, every group of experiment repeats detection 3 times；Simultaneously to be not added with the cell killing efficiency of any antibody as emptyWhite control.

As a result as shown in figure 13：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any resistUnder conditions of body, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell increases significantly to the killing-efficiency of Raji cells, wherein CD19-CD3-The Cell killing efficacy that GITR TsAb_D are mediated is best, and killing-efficiency respectively may be about 93%, 77% and 73%, CD19-CD3-The effect of GITR TsAb_M is taken second place, and killing-efficiency is about 88%, 83% and the effect of 66%, CD19-CD3 BsAb are most weak, is killedHinder efficiency and respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml), CD19-The CIK cell that CD3-GITR TsAb_D and CD19-CD3-GITR TsAb_M are mediated still has the killing-efficiency of Raji cellsIt improves to a certain extent, killing-efficiency respectively may be about 57% and 49%, and CD19-CD3 BsAb do not have substantially compared with blank controlIt is effective.The above results illustrate T cell that tri- specific antibodies of CD19-CD3-GITR TiTE of two kinds of forms are mediated to CD19The target killing activity of positive tumor cell is superior to CD19-CD3 BiTE bispecific antibodies, and wherein dimeric forms are more singleBody form has better effect.

Embodiment 17：The structure of CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D carrier for expression of eukaryonIt builds

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules CD40L albumen is named as CD19-CD3-CD40L TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-CD40L TsAb_M and the design of CD19-CD3-CD40L TsAb_D constructing plans

The specific constructing plans of CD19-CD3-CD40L TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-CD 40 L scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, then pass through junction fragment 2 between AntiCD3 McAb scFv and anti-CD 40 L scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-CD40L TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAbScFv is connected with the sequence of anti-CD 40 L scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAbIt is connected between scFv by junction fragment 1 (Linker 1), with IgD hinges between AntiCD3 McAb scFv and anti-CD 40 L scFv sequencesArea (Ala 90-Val 170) is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, resistCD40L scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65.

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66.

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68.

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69.

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of anti-CD 40 L scFv is as shown in SEQ ID NO.83, specially：

GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-CD 40 L scFv is as shown in SEQ ID NO.84, specially：

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of anti-CD 40 L scFv is as shown in SEQ ID NO.82, specially：

GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence such as SEQ of the CD19-CD3-CD40L TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in ID NO.2.

The nucleotide sequence such as SEQ of the CD19-CD3-CD40L TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in ID NO.4.

The nucleotide sequence such as SEQ of the CD19-CD3-CD40L TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6.

The nucleotide sequence such as SEQ of the CD19-CD3-CD40L TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8.

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89.

2nd, CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1(being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the three-specific antibody of dimeric forms, separately designPrimer as shown in table 5, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplificationZhou Hongxun Science and Technology Ltd.s synthesize.

It is built for the clone of CD19-CD3-CD40L TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- CD40L-F and pcDNA3.1-CD40L-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of Linker 2+ anti-CD 40 Ls scFv；For the clone of CD19-CD3-CD40L TsAb_DStructure, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, is then utilized respectively primerSig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD40L-F andPcDNA3.1-CD40L-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker2nd, the gene order of anti-CD 40 L scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from WuJiang Jinan protein Science and Technology Ltd.) splicing monomer and dimeric forms three-specific antibody full-length gene order have no respectivelySeam is cloned on the pcDNA3.1 expression vectors through EcoRI and HindIII linearization process, converts bacillus coli DH 5 alpha, is utilizedBacterium colony PCR carries out positive clone identification, is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.It will then be sequenced justTrue recon (recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-CD40L TsAb_M of monomeric form and the CD19-CD3- of dimeric formsThe full-length gene order of CD40L TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-CD40L TsAb_M of monomeric form is as shown in SEQ ID NO.33,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The nucleotide sequence of the CD19-CD3-CD40L TsAb_D of dimeric forms is as shown in SEQ ID NO.35, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCAAGAGCTACGGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGC。

The primer used in table 5.CD19-CD3-CD40L three-specific antibody gene clonings

Embodiment 18：The expression and purification of CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D

First, the expression of CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40LTsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombination matter in Example 17Grain：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollect culture supernatant and carry out destination protein detection of expression

2nd, the purifying of CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in figure 14 under reduction and non reducing conditions.It can be seen from the figure that through the affine layers of Protein LAfter analysing column purification, the purity of CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D recombinant proteins is equal>95%：Wherein the theoretical molecular weight of CD19-CD3-CD40L TsAb_M recombinant proteins is 79.2kDa, and reduction and non reducing conditions descend the eggSingle electrophoretic band is presented in Bai Jun, and molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Figure 14 A)；The theoretical molecular weight of CD19-CD3-CD40L TsAb_D recombinant proteins is 87.1kDa, the protein electrophoresis band institute under reducing conditionPresentation molecular weight is consistent with monomer, and it is consistent with dimer (about 180kDa) that molecular weight is presented in electrophoretic band under non reducing conditions(Figure 14 B) illustrates that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibodyFor dimeric forms.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-CD40L TsAb_M is monomeric form, and CD19-CD3-CD40L TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.32 of the CD19-CD3-CD40L TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

The amino acid sequence of the CD19-CD3-CD40L TsAb_D of dimeric forms is as shown in SEQ ID NO.34, specificallyFor：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40.

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41.

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42.

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45.

The amino acid sequence of anti-CD 40 L scFv is as shown in SEQ ID NO.58, specially：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

The amino acid sequence of the heavy chain variable region of anti-CD 40 L scFv is as shown in SEQ ID NO.59, specially：

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-CD 40 L scFv is as shown in SEQ ID NO.60, specially：

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKR。

The amino acid sequence such as SEQ of the CD19-CD3-CD40L TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in ID NO.1.

The amino acid sequence such as SEQ of the CD19-CD3-CD40L TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in ID NO.3.

The amino acid sequence such as SEQ of the CD19-CD3-CD40L TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5.

The amino acid sequence such as SEQ of the CD19-CD3-CD40L TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7.

Embodiment 19：The antigen of ELISA detection CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_DWith reference to activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind CD40L-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-CD40L TsAb_M or CD19-CD3-CD40L purified with 10 μ g/mlTsAb_D carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as shown in fig. 15 a and fig. 15b：Figure 15 A illustrate CD19-CD3-CD40L TsAb_M and recombinant antigenCD19-hFc, CD3-hFc and CD40L-hFc are respectively provided with external combination activity, and wherein CD40L combines active highest, and CD19 is combinedActivity is taken second place, and it is weaker that CD3 combines activity；Figure 15 B illustrate CD19-CD3-CD40L TsAb_D and recombinant antigen CD19-hFc,CD3-hFc and CD40L-hFc equally has external combination activity, and wherein CD40L combines active highest, and CD19 is combined active timeIt, it is weaker that CD3 combines activity.

Embodiment 20：The cell killing experiment of CD19-CD3-CD40L three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-CD40L TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-CD40L TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-CD40L TsAb_M and CD19-CD3-CD40L TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of trainings altogetherAfter supporting 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to following public affairsFormula calculates cell killing efficiency, and every group of experiment repeats detection 3 times；Simultaneously using be not added with the cell killing efficiency of any antibody asBlank control.

As a result as shown in figure 16：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any resistUnder conditions of body, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell significantly increases the killing-efficiency of Raji cells, wherein CD19-CD3-The Cell killing efficacy that CD40L TsAb_D are mediated is best, and killing-efficiency respectively may be about 94%, 90% and 82%, CD19-The effect of CD3-CD40L TsAb_M is taken second place, killing-efficiency be about 91%, 88% and the effect of 73%, CD19-CD3BsAb mostWeak, killing-efficiency respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml),The CIK cell that CD19-CD3-CD40L TsAb_D and CD19-CD3-CD40L TsAb_M are mediated imitates the killing of Raji cellsRate still has to be improved to a certain extent, and killing-efficiency respectively may be about 68% and 61%, and CD19-CD3BsAb is compared with blank controlSubstantially there is no effect.The above results illustrate the T cell that tri- specific antibodies of CD19-CD3-CD40L TiTE of two kinds of forms are mediatedCD19-CD3 BiTE bispecific antibodies, wherein the dimerization bodily form are superior to the target killing activity of CD19 positive tumor cellsFormula has better effect compared with monomeric form.

Embodiment 21：The structure of CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D carrier for expression of eukaryon

In the present invention, with mankind's CD19 albumen on lymthoma B cell surface, T cell surface mankind CD3 and T cell are justCostimulatory molecules CD27 albumen is named as CD19-CD3-CD27 TsAb for the TiTE three-specific antibodies of target spot.

First, CD19-CD3-CD27 TsAb_M and the design of CD19-CD3-CD27 TsAb_D constructing plans

The specific constructing plans of CD19-CD3-CD27 TsAb_M of monomeric form are：Anti- CD19 scFv, AntiCD3 McAb scFv andThe sequence of anti-CD27 scFv is connected by junction fragment (Linker), specifically, between anti-CD19 scFv and AntiCD3 McAb scFvIt is connected by junction fragment 1 (Linker 1), then passes through junction fragment 2 between AntiCD3 McAb scFv and anti-CD27 scFv sequences(Linker 2) is connected.

The specific constructing plans of CD19-CD3-CD27 TsAb_D of dimeric forms are：Anti- CD19 scFv, AntiCD3 McAb scFvBe connected with the sequence of anti-CD27 scFv by junction fragment (Linker), specifically, anti-CD19 scFv and AntiCD3 McAb scFv itBetween by junction fragment 1 (Linker 1) be connected, with IgD hinge areas (Ala between AntiCD3 McAb scFv and anti-CD27 scFv sequences90-Val 170) it is connected as junction fragment 2 (Linker 2).

For three-specific antibody is made to be expressed in mammalian cell, for anti-CD19 scFv, AntiCD3 McAb scFv, resistCD27 scFv sequences have carried out the codon optimization of lactation system expression.

Specifically, the nucleotide sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.65.

The nucleotide sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.66.

The nucleotide sequence of anti-CD19 scFv is as shown in SEQ ID NO.64.

The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.68.

The nucleotide sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.69.

The nucleotide sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.67.

The nucleotide sequence of the heavy chain variable region of anti-CD27 scFv is as shown in SEQ ID NO.86, specially：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC。

The nucleotide sequence of the light chain variable region of anti-CD27 scFv is as shown in SEQ ID NO.87, specially：

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence of anti-CD27 scFv is as shown in SEQ ID NO.85, specially：

CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence such as SEQ ID of the CD19-CD3-CD27 TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.2.

The nucleotide sequence such as SEQ ID of the CD19-CD3-CD27 TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.4.

The nucleotide sequence such as SEQ of the CD19-CD3-CD27 TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.6.

The nucleotide sequence such as SEQ of the CD19-CD3-CD27 TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.8.

For three-specific antibody is made to be expressed in CHO-S cells and in successful secretion to culture medium, has selected antibody-secretingThe signal peptide of type expression is used for this embodiment.

The amino acid sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.88.

The nucleotide sequence of the secreting, expressing signal peptide is as shown in SEQ ID NO.89.

2nd, CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D construction of eukaryotic expression vector

The construction and expression of three-specific antibody of the present invention selects mammalian cell albumen transient expression vector pcDNA3.1(being purchased from Shanghai Ying Jun bio tech ltd).For structure monomer and the three-specific antibody of dimeric forms, separately designPrimer as shown in table 6, all primers are synthesized by Suzhou Jin Weizhi bio tech ltd, and gene template is by reviving needed for amplificationZhou Hongxun Science and Technology Ltd.s synthesize.

It is built for the clone of CD19-CD3-CD27 TsAb_M, first using primer pcDNA3.1-Sig-F and Sig-RAmplify signal peptide fragment, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R,CD3-(GGGGS)₃- CD27-F and pcDNA3.1-CD27-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAbsscFv、(GGGGS)₃The gene order of the anti-CD27 scFv of Linker 2+；For clone's structure of CD19-CD3-CD27 TsAb_DIt builds, equally amplifies signal peptide fragment using primer pcDNA3.1-Sig-F and Sig-R first, be then utilized respectively primer Sig-CD19-F and CD19-R, CD19-G4S-CD3-F and CD3-R, CD3-IgD-F and IgD-R, IgD-CD27-F and pcDNA3.1-CD27-R amplifies anti-CD19 scFv, GGGGS Linker 1+ AntiCD3 McAb scFv, IgD hinge areas Linker 2, anti-CD27The gene order of scFv.After amplification, utilizeMono- step directed cloning kits of PCR (are purchased from Wujiang offshore albumenMatter Science and Technology Ltd.) splice respectively monomer and dimeric forms three-specific antibody full-length gene order and it is seamless be cloned into throughOn the pcDNA3.1 expression vectors of EcoRI and HindIII linearization process, bacillus coli DH 5 alpha is converted, is carried out using bacterium colony PCRPositive clone identification is accredited as positive recon (recombinant plasmid) and carries out sequencing identification.Correct recon will then be sequenced(recombinant plasmid) arranges to take out in plasmid, for the transfection of CHO-S cells.

Know through sequencing, the CD19-CD3-CD27 TsAb_M of monomeric form and the CD19-CD3- of dimeric formsThe full-length gene order of CD27TsAb_D is correct, is consistent with expection.

Specifically, the nucleotide sequence of the CD19-CD3-CD27 TsAb_M of monomeric form is as shown in SEQ ID NO.37,Specially：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGGAGGCGGAGGTTCCGGCGGTGGGGGATCGGGGGGTGGAGGGAGTCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The nucleotide sequence of the CD19-CD3-CD27 TsAb_D of dimeric forms is as shown in SEQ ID NO.39, specificallyFor：

GACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGCGCCCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGCGGAGGGTCCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCCGCCCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCCGCTACACCATGCACTGGGTGAAGCAGCGCCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCCGCGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGCTGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACCGCTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGGCCAGCAAGAGCAAGAAGGAGATCTTCCGCTGGCCCGAGAGCCCCAAGGCCCAGGCCAGCAGCGTGCCCACCGCCCAGCCCCAGGCCGAGGGCAGCCTGGCCAAGGCCACCACCGCCCCCGCCACCACCCGCAACACCGGCCGCGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGAAGGAGGAGCAGGAGGAGCGCGAGACCAAGACCCCCGAGTGCCCCAGCCACACCCAGCCCCTGGGCGTGCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTACGACGGCAGCAACAAGTACTACGCCGACAGCGTGAAGGGCCGCTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGGCAGCGGCAACTGGGGCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGCGTGACCATCACCTGCCGCGCCAGCCAGGGCATCAGCCGCTGGCTGGCCTGGTACCAGCAGAAGCCCGAGAAGGCCCCCAAGAGCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACACCTACCCCCGCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG。

The primer used in table 6.CD19-CD3-CD27 three-specific antibody gene clonings

Embodiment 22：The expression and purification of CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D

First, the expression of CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D

1.1.CHO-S 1 day passage density is 0.5 before cell (being purchased from Thermo Fisher Scientific companies) transfection~0.6 × 10⁶/ml；

1.2. the transfection same day carries out cell density statistics, when density is 1~1.4 × 10⁶/ ml, vigor>When 90%, it can useIn plasmid transfection；

1.3. transfection composite is prepared：Each project (CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D two centrifuge tube/culture bottles) need to be prepared, by taking 20ml as an example, placed respectively, prepared recombinant plasmid in Example 21：

Manage 1. 600 μ l PBS of middle addition, 20 μ g recombinant plasmids, mixing；

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

1.4. it by the transfection reagent after dilution, adds in the recombinant plasmid to after diluting, is uniformly mixed, it is multiple to be configured to transfectionClose object；

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

1.6. in 37 DEG C, CO₂Concentration 8% carries out Transfected cells culture under the conditions of shaking speed 130rpm, is received after 5 daysCollect culture supernatant and carry out destination protein detection of expression

2nd, the purifying of CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D

2.1 sample pretreatment

Above-mentioned Transfected cells culture supernatant 20ml is taken, buffer solution 20mM PB, 200mM NaCl is added in and adjusts pH to 7.5；

2.2Protein L affinity chromatography column purifications

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

Buffer solution A (Buffer A)：PBS, pH7.4

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

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

Purification process：Using 100 type protein purification systems of AKTA explorer (be purchased from GE Healthcare companies),Protein L affinity columns are pre-processed with Buffer A, take culture supernatant loading, collect efflux.After loading, with extremelyFew 1.5ml Buffer A balance chromatographic column, are eluted respectively with Buffer B and Buffer C after balance, collect destination protein and wash(collecting pipe of eluent needs to be previously added 1% 1M Tris, pH8.0 to neutralize eluent pH value, Tris final concentrations de- liquidAbout 10mM), in finally concentration dialysis to buffer solution PBS.

CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 the TsAb_D recombinant proteins finally purified are through SDS-PAGE is analyzed, and electrophoretogram is as shown in figure 17 under reduction and non reducing conditions.It can be seen from the figure that through the affine layers of Protein LAfter analysing column purification, the purity of CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D recombinant proteins is equal>95%：ItsThe theoretical molecular weight of middle CD19-CD3-CD27 TsAb_M recombinant proteins is 80.1kDa, reduction and the albumen under non reducing conditionsSingle electrophoretic band is presented, molecular weight is consistent with monomer, therefore the three-specific antibody is monomeric form (Figure 17 A)；CD19-The theoretical molecular weight of CD3-CD27 TsAb_D recombinant proteins is 88.0kDa, and the protein electrophoresis band is presented point under reducing conditionSon amount is consistent with monomer, and molecular weight (about 180kDa) consistent with dimer (Figure 17 B) is presented in electrophoretic band under non reducing conditions,Illustrate that two protein moleculars can form disulfide bond by IgD hinge areas and be connected with each other, therefore the three-specific antibody is dimerForm.

In addition, the recombinant protein sample of purifying is through N/C terminal sequence analysis, the results showed that expressed recombinant protein sampleEqual frame is errorless, and consistent with theoretical N/C terminal amino acid sequences, mass spectral analysis further confirms that CD19-CD3-CD27 TsAb_MFor monomeric form, CD19-CD3-CD27 TsAb_D are dimeric forms.

Therefore, it can be seen that, the amino acid sequence such as SEQ ID NO.36 of the CD19-CD3-CD27 TsAb_M of monomeric formIt is shown, specially：

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

The amino acid sequence of the CD19-CD3-CD27 TsAb_D of dimeric forms is as shown in SEQ ID NO.38, specificallyFor：DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

The amino acid sequence of anti-CD19 scFv is as shown in SEQ ID NO.40.

The amino acid sequence of the heavy chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.41.

The amino acid sequence of the light chain variable region of anti-CD19 scFv is as shown in SEQ ID NO.42.

The amino acid sequence of AntiCD3 McAb scFv is as shown in SEQ ID NO.43.

The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.44.

The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv is as shown in SEQ ID NO.45.

The amino acid sequence of anti-CD27 scFv is as shown in SEQ ID NO.61, specially：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

The amino acid sequence of the heavy chain variable region of anti-CD27 scFv is as shown in SEQ ID NO.62, specially：

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSS。

The amino acid sequence of the light chain variable region of anti-CD27 scFv is as shown in SEQ ID NO.63, specially：

DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK。

The amino acid sequence such as SEQ ID of the CD19-CD3-CD27 TsAb_M junction fragments 1 (Linker 1) of monomeric formShown in NO.1.

The amino acid sequence such as SEQ ID of the CD19-CD3-CD27 TsAb_M junction fragments 2 (Linker 2) of monomeric formShown in NO.3.

The amino acid sequence such as SEQ of the CD19-CD3-CD27 TsAb_D junction fragments 1 (Linker 1) of dimeric formsShown in ID NO.5.

The amino acid sequence such as SEQ of the CD19-CD3-CD27 TsAb_D junction fragments 2 (Linker 2) of dimeric formsShown in ID NO.7.

Embodiment 23：The antigen knot of ELISA detection CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_DClose activity

ELISA operating procedures：

1. recombinant antigen is coated with：Mankind CD19-hFc, mankind CD3-hFc and mankind CD27-hFc fusion proteins (are purchased from WuJiang Jinan protein Science and Technology Ltd.) it is coated with 96 orifice plates respectively, antigen concentration is 1 μ g/ml, and coating volume is 100 μ l/ holes,Coating condition is stayed overnight for 1 hour or 4 DEG C for 37 DEG C, and the formula of coating buffer solution (PBS) is：3.58g Na₂HPO₄, 0.24gNaH₂PO₄, 0.2g KCl, 8.2g NaCl, 950ml H₂O, with 1mol/L HCl or 1mol/L NaOH tune pH to 7.4, moisturizing is extremely1L；

2. closing：After PBS board-washings 4 times, confining liquid PBSA (PBS+2%BSA (V/W)), 200 μ l/ holes are added in.37 DEG C of closings1 hour；

3. sample-adding：After PBS board-washings 4 times, the three-specific antibody sample of purifying is separately added into, 100 μ l/ holes, 37 DEG C are incubated 1Hour, sample gradient preparation method：The CD19-CD3-CD27 TsAb_M or CD19-CD3-CD27TsAb_ purified with 10 μ g/mlD carries out 6 gradients of doubling dilution, each gradient sets 2 multiple holes as initial concentration；

4. colour developing：It is dilute by 1/5000 with confining liquid PBSA after PBST (PBS+0.05%Tween-20 (V/V)) board-washing 4 timesThe colour developing antibody (purchased from Abcam companies) of HRP labels is released, is added in by 100 μ l/ holes, 37 DEG C are incubated 1 hour.After PBS board-washings 4 times,Developing solution TMB (being purchased from KPL companies) is added, 100 μ l/ holes, room temperature is protected from light colour developing 5~10 minutes；

5. it terminates reaction to measure with result：Add terminate liquid (1M HCl), 100 μ l/ holes, the 450nm wavelength in microplate readerLower reading light absorption value (OD₄₅₀)。

ELISA results are as shown in Figure 18 A and Figure 18 B：Figure 18 A illustrate CD19-CD3-CD27 TsAb_M and recombinant antigenCD19-hFc, CD3-hFc and CD27-hFc are respectively provided with external combination activity, and wherein CD27 combines active highest, and CD19 combines workProperty take second place, CD3 combine activity it is weaker；Figure 18 B illustrate CD19-CD3-CD27 TsAb_D and recombinant antigen CD19-hFc, CD3-HFc and CD27-hFc equally has external combination activity, and wherein CD27 combines active highest, and CD19 combination activity is taken second place, CD3 knotsIt is weaker to close activity.

Embodiment 24：The cell killing experiment of CD19-CD3-CD27 three-specific antibodies mediation

It is experiment material with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC)Material, with tri- specific antibodies of TiTE (CD19-CD3-CD27 TsAb_M), the dimer of the above-mentioned monomeric form prepared by the present inventionTri- specific antibodies of TiTE (CD19-CD3-CD27 TsAb_D) and anti-CD19/ AntiCD3 McAbs BiTE bispecific antibodies of form(CD19-CD3 BsAb, purchased from Wujiang Alongshore Protein Technology Co., Ltd.) is respectively acting on people's blood of same donor sourceCIK cell (CD3 prepared by PBMC⁺CD56⁺) and CCL-86Raji lymphoma cells (CD19⁺, purchased from ATCC), detection cell is deadSituation is died, compares killing-efficiency difference of three kinds of antibody-mediated CIK effector cells to CCL-86Raji target cells.

Cell killing experimental procedure：

The separation of 1.PBMC：Using the volunteer's anticoagulated blood newly extracted, add in isometric medical saline, along fromHeart tube wall is slowly added to the lymphocyte separation medium isometric with blood (purchased from GE Healthcare companies), keeps liquid level pointLayer is apparent, and 2000rpm centrifugation 20min draw the cellular layer of intermediate white haze shape in new centrifuge tube, add in 2 times or more volumePBS buffer solution washing, 1100rpm centrifugation 10min, repeated washing is primary, with 15 free serum cultures of X-vivo being pre-chilled on a small quantityBase (being purchased from Lonza companies) is resuspended, and cell count is for use；

2.CIK cell culture and amplification：PBMC CIK basal mediums (90%X-vivo15+10%FBS) (are purchased fromGbico companies) it is resuspended, adjustment cell density is 1 × 10⁶/ ml is added to full length antibody Anti-CD3 (5ug/ml), overall length resistsIn body Anti-CD28 (5ug/ml) and the coated T25 culture bottles of NovoNectin (25ug/ml) (full length antibody withNovoNectin is purchased from Wujiang Alongshore Protein Technology Co., Ltd.), at the same add cell factor 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 technologyDepartment), incubator is placed in, in saturated humidity, 37 DEG C, 5.0%CO₂Under conditions of cultivated.After overnight incubation, 500U/ is addedThe IL-2 (be purchased from Wujiang Alongshore Protein Technology Co., Ltd.) of ml continues to cultivate, and counts within every 2~3 days and with adding 500U/mlThe CIK basal mediums of IL-2 press 1 × 10⁶The density of/ml carries out cell passage；

3.CIK cells are to the killing-efficiency of Raji cells：Cell killing experiment is carried out in 96 orifice plates, reaction volume is100uL takes the CIK cell 1 × 10 of above-mentioned culture⁵It is a, add in Raji cells 1 × 10⁵A (CIK effector cell：Raji target cells(E：T ratios) it is 1：1), add respectively different final concentrations (25,12.5,6.25,3.125ng/ml) CD19-CD3 BsAb,CD19-CD3-CD27 TsAb_M and CD19-CD3-CD27 TsAb_D antibody samples, 3~5min of room temperature mixing, 37 DEG C of co-cultivationsAfter 3h, the CCK-8 of 10 μ l is added per hole, 37 DEG C of the reaction was continued 2~3h then survey OD with microplate reader₄₅₀Value, according to the following formulaCell killing efficiency is calculated, every group of experiment repeats detection 3 times；Simultaneously to be not added with the cell killing efficiency of any antibody as emptyWhite control.

As a result as shown in figure 19：As CIK effector cell：Raji target cells (E：T ratios) it is 1：When 1, it is being not added with any resistUnder conditions of body, CIK cell is about 23% to the killing-efficiency of Raji cells 3h；Addition higher concentration antibody (25,12.5,Under conditions of 6.25ng/ml), CIK cell significantly increases the killing-efficiency of Raji cells, wherein CD19-CD3-The Cell killing efficacy that CD27 TsAb_D are mediated is best, and killing-efficiency respectively may be about 89%, 84% and 74%, CD19-CD3-The effect of CD27 TsAb_M is taken second place, and killing-efficiency is about 89%, 84% and the effect of 67%, CD19-CD3 BsAb are most weak, is killedHinder efficiency and respectively may be about 80%, 54% and 54%；Under conditions of addition low concentration antibody (3.125ng/ml), CD19-The CIK cell that CD3-CD27 TsAb_D and CD19-CD3-CD27 TsAb_M are mediated still has the killing-efficiency of Raji cellsIt improves to a certain extent, killing-efficiency respectively may be about 55% and 49%, and CD19-CD3 BsAb do not have substantially compared with blank controlIt is effective.The above results illustrate T cell that tri- specific antibodies of CD19-CD3-CD27 TiTE of two kinds of forms are mediated to CD19The target killing activity of positive tumor cell is superior to CD19-CD3 BiTE bispecific antibodies, and wherein dimeric forms are more singleBody form has better effect.

The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation,It should be pointed out that for those skilled in the art, under the premise of the method for the present invention is not departed from, can also makeSeveral improvement and supplement, these are improved 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 made using disclosed above technology contents it is a little moreDynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present inventionThe variation, modification and evolution of any equivalent variations that above-described embodiment is made still fall within the range of technical scheme of the present inventionIt is interior.

SEQUENCE LISTING

<110>Shanghai offshore bio tech ltd

<120>Three functional moleculars and its application of a kind of combination CD19, CD3 and the positive costimulatory molecules of T cell

<130> 164332

<160> 115

<170> PatentIn version 3.3

<210> 1

<211> 5

<212> PRT

<213> Artificial

<220>

<223>Junction fragment 1 in positive three specific antibody of costimulatory molecules of anti-CD19/ AntiCD3 McAbs/anti- T cell of monomeric form

Amino acid sequence

<400> 1

Gly Gly Gly Gly Ser

1 5

<210> 2

<211> 15

<212> DNA

<213> Artificial

<220>

<223>Junction fragment 1 in positive three specific antibody of costimulatory molecules of anti-CD19/ AntiCD3 McAbs/anti- T cell of monomeric form

Nucleotide sequence

<400> 2

ggtggcggag ggtcc 15

<210> 3

<211> 15

<212> PRT

<213> Artificial

<220>

<223>Junction fragment 2 in positive three specific antibody of costimulatory molecules of anti-CD19/ AntiCD3 McAbs/anti- T cell of monomeric form

Amino acid sequence

<400> 3

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

1 5 10 15

<210> 4

<211> 45

<212> DNA

<213> Artificial

<220>

<223>Junction fragment 2 in positive three specific antibody of costimulatory molecules of anti-CD19/ AntiCD3 McAbs/anti- T cell of monomeric form

Nucleotide sequence

<400> 4

ggaggcggag gttccggcgg tgggggatcg gggggtggag ggagt 45

<210> 5

<211> 5

<212> PRT

<213> Artificial

<220>

<223>Junction fragment in the positive costimulatory molecules three-specific antibody of anti-CD19/ AntiCD3 McAbs/anti- T cell of dimeric forms

1 amino acid sequence

<400> 5

Gly Gly Gly Gly Ser

1 5

<210> 6

<211> 15

<212> DNA

<213> Artificial

<220>

<223>Junction fragment in the positive costimulatory molecules three-specific antibody of anti-CD19/ AntiCD3 McAbs/anti- T cell of dimeric forms

1 nucleotide sequence

<400> 6

ggtggcggag ggtcc 15

<210> 7

<211> 81

<212> PRT

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<220>

<223>Junction fragment in the positive costimulatory molecules three-specific antibody of anti-CD19/ AntiCD3 McAbs/anti- T cell of dimeric forms

2 amino acid sequence

<400> 7

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> 8

<211> 243

<212> DNA

<213> Artificial

<220>

<223>Junction fragment in the positive costimulatory molecules three-specific antibody of anti-CD19/ AntiCD3 McAbs/anti- T cell of dimeric forms

2 nucleotide sequence

<400> 8

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> 9

<211> 134

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind CD28 extracellular regions of T cell

<400> 9

Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn

1 5 10 15

Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu

20 25 30

Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys

35 40 45

Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr

50 55 60

Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr

65 70 75 80

Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile

85 90 95

Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly

100 105 110

Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe

115 120 125

Pro Gly Pro Ser Lys Pro

130

<210> 10

<211> 163

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind 4-1BB extracellular regions of T cell

<400> 10

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln

<210> 11

<211> 120

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind ICOS extracellular regions of T cell

<400> 11

Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile Phe His Asn Gly

1 5 10 15

Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe Lys

20 25 30

Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys Thr

35 40 45

Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys Phe Cys His

50 55 60

Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp

65 70 75 80

His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser Ile Phe Asp Pro

85 90 95

Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu His Ile Tyr Glu

100 105 110

Ser Gln Leu Cys Cys Gln Leu Lys

115 120

<210> 12

<211> 186

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind OX40 extracellular regions of T cell

<400> 12

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1 5 10 15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

20 25 30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

35 40 45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

50 55 60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65 70 75 80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

85 90 95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

100 105 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

115 120 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

130 135 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145 150 155 160

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

165 170 175

Arg Pro Val Glu Val Pro Gly Gly Arg Ala

180 185

<210> 13

<211> 137

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind GITR extracellular regions of T cell

<400> 13

Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg Leu Leu Leu

1 5 10 15

Gly Thr Gly Thr Asp Ala Arg Cys Cys Arg Val His Thr Thr Arg Cys

20 25 30

Cys Arg Asp Tyr Pro Gly Glu Glu Cys Cys Ser Glu Trp Asp Cys Met

35 40 45

Cys Val Gln Pro Glu Phe His Cys Gly Asp Pro Cys Cys Thr Thr Cys

50 55 60

Arg His His Pro Cys Pro Pro Gly Gln Gly Val Gln Ser Gln Gly Lys

65 70 75 80

Phe Ser Phe Gly Phe Gln Cys Ile Asp Cys Ala Ser Gly Thr Phe Ser

85 90 95

Gly Gly His Glu Gly His Cys Lys Pro Trp Thr Asp Cys Thr Gln Phe

100 105 110

Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys

115 120 125

Val Pro Gly Ser Pro Pro Ala Glu Pro

130 135

<210> 14

<211> 215

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind CD40L extracellular regions of T cell

<400> 14

His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp

1 5 10 15

Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser

20 25 30

Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe

35 40 45

Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser

50 55 60

Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val

65 70 75 80

Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu

85 90 95

Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly

100 105 110

Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln

115 120 125

Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile

130 135 140

Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu

145 150 155 160

Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser

165 170 175

Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe

180 185 190

Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr

195 200 205

Ser Phe Gly Leu Leu Lys Leu

210 215

<210> 15

<211> 172

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the positive costimulatory molecules mankind CD27 extracellular regions of T cell

<400> 15

Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln

1 5 10 15

Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys

20 25 30

Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro

35 40 45

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

50 55 60

Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr

65 70 75 80

Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys

85 90 95

Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg

100 105 110

Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr

115 120 125

Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu

130 135 140

Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro

145 150 155 160

Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg

165 170

<210> 16

<211> 759

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-4-1BB TsAb_M of monomeric form

<400> 16

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 Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser

515 520 525

Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly

530 535 540

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

545 550 555 560

Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu

565 570 575

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

580 585 590

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

595 600 605

Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp

610 615 620

Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

625 630 635 640

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

645 650 655

Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

660 665 670

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys

675 680 685

Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala

690 695 700

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

705 710 715 720

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

725 730 735

Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Cys Gly Gly

740 745 750

Thr Lys Val Glu Ile Lys Arg

755

<210> 17

<211> 2277

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-4-1BB TsAb_M of monomeric form

<400> 17

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 gcagcagtgg 1560

ggcgccggcc tgctgaagcc cagcgagacc ctgagcctga cctgcgccgt gtacggcggc 1620

agcttcagcg gctactactg gagctggatc cgccagagcc ccgagaaggg cctggagtgg 1680

atcggcgaga tcaaccacgg cggctacgtg acctacaacc ccagcctgga gagccgcgtg 1740

accatcagcg tggacaccag caagaaccag ttcagcctga agctgagcag cgtgaccgcc 1800

gccgacaccg ccgtgtacta ctgcgcccgc gactacggcc ccggcaacta cgactggtac 1860

ttcgacctgt ggggccgcgg caccctggtg accgtgagca gcggcggcgg cggcagcggc 1920

ggcggcggca gcggcggcgg cggcagcgag atcgtgctga cccagagccc cgccaccctg 1980

agcctgagcc ccggcgagcg cgccaccctg agctgccgcg ccagccagag cgtgagcagc 2040

tacctggcct ggtaccagca gaagcccggc caggcccccc gcctgctgat ctacgacgcc 2100

agcaaccgcg ccaccggcat ccccgcccgc ttcagcggca gcggcagcgg caccgacttc 2160

accctgacca tcagcagcct ggagcccgag gacttcgccg tgtactactg ccagcagcgc 2220

agcaactggc cccccgccct gaccttctgc ggcggcacca aggtggagat caagcgc 2277

<210> 18

<211> 825

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-4-1BB TsAb_D of dimeric forms

<400> 18

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 Gln Gln Trp Gly Ala Gly Leu Leu Lys

580 585 590

Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp

675 680 685

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

690 695 700

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

705 710 715 720

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

725 730 735

Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln

740 745 750

Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn

755 760 765

Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

770 775 780

Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val

785 790 795 800

Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Cys

805 810 815

Gly Gly Thr Lys Val Glu Ile Lys Arg

820 825

<210> 19

<211> 2475

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-4-1BB TsAb_D of dimeric forms

<400> 19

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

gtgcagctgc agcagtgggg cgccggcctg ctgaagccca gcgagaccct gagcctgacc 1800

tgcgccgtgt acggcggcag cttcagcggc tactactgga gctggatccg ccagagcccc 1860

gagaagggcc tggagtggat cggcgagatc aaccacggcg gctacgtgac ctacaacccc 1920

agcctggaga gccgcgtgac catcagcgtg gacaccagca agaaccagtt cagcctgaag 1980

ctgagcagcg tgaccgccgc cgacaccgcc gtgtactact gcgcccgcga ctacggcccc 2040

ggcaactacg actggtactt cgacctgtgg ggccgcggca ccctggtgac cgtgagcagc 2100

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgagat cgtgctgacc 2160

cagagccccg ccaccctgag cctgagcccc ggcgagcgcg ccaccctgag ctgccgcgcc 2220

agccagagcg tgagcagcta cctggcctgg taccagcaga agcccggcca ggccccccgc 2280

ctgctgatct acgacgccag caaccgcgcc accggcatcc ccgcccgctt cagcggcagc 2340

ggcagcggca ccgacttcac cctgaccatc agcagcctgg agcccgagga cttcgccgtg 2400

tactactgcc agcagcgcag caactggccc cccgccctga ccttctgcgg cggcaccaag 2460

gtggagatca agcgc 2475

<210> 20

<211> 760

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-ICOS TsAb_M of monomeric form

<400> 20

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 Gly

530 535 540

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

545 550 555 560

Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys

565 570 575

Phe Gln Gly Arg Val Thr Met Thr Arg 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 Tyr

595 600 605

Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala

610 615 620

Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

645 650 655

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

660 665 670

Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu Ala Trp

675 680 685

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Val Ala

690 695 700

Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

705 710 715 720

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

725 730 735

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

740 745 750

Gln Gly Thr Lys Val Glu Ile Lys

755 760

<210> 21

<211> 2280

<212> DNA

<213> Artificial

<220>

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

<400> 21

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

accttcaccg gctactacat gcactgggtg cgccaggccc ccggccaggg cctggagtgg 1680

atgggctgga tcaaccccca cagcggcggc accaactacg cccagaagtt ccagggccgc 1740

gtgaccatga cccgcgacac cagcatcagc accgcctaca tggagctgag ccgcctgcgc 1800

agcgacgaca ccgccgtgta ctactgcgcc cgcacctact actacgacag cagcggctac 1860

taccacgacg ccttcgacat ctggggccag ggcaccatgg tgaccgtgag cagcggcggc 1920

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 1980

cccagcagcg tgagcgccag cgtgggcgac cgcgtgacca tcacctgccg cgccagccag 2040

ggcatcagcc gcctgctggc ctggtaccag cagaagcccg gcaaggcccc caagctgctg 2100

atctacgtgg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2160

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2220

tgccagcagg ccaacagctt cccctggacc ttcggccagg gcaccaaggt ggagatcaag 2280

<210> 22

<211> 826

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-ICOS TsAb_D of dimeric forms

<400> 22

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 Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

610 615 620

Glu Trp Met Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala

625 630 635 640

Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg 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 Tyr Cys Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His

675 680 685

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

690 695 700

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

705 710 715 720

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

725 730 735

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

820 825

<210> 23

<211> 2478

<212> DNA

<213> Artificial

<220>

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

<400> 23

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 cttcaccggc tactacatgc actgggtgcg ccaggccccc 1860

ggccagggcc tggagtggat gggctggatc aacccccaca gcggcggcac caactacgcc 1920

cagaagttcc agggccgcgt gaccatgacc cgcgacacca gcatcagcac cgcctacatg 1980

gagctgagcc gcctgcgcag cgacgacacc gccgtgtact actgcgcccg cacctactac 2040

tacgacagca gcggctacta ccacgacgcc ttcgacatct ggggccaggg caccatggtg 2100

accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 2160

atccagatga cccagagccc cagcagcgtg agcgccagcg tgggcgaccg cgtgaccatc 2220

acctgccgcg ccagccaggg catcagccgc ctgctggcct ggtaccagca gaagcccggc 2280

aaggccccca agctgctgat ctacgtggcc agcagcctgc agagcggcgt gcccagccgc 2340

ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcagcccgag 2400

gacttcgcca cctactactg ccagcaggcc aacagcttcc cctggacctt cggccagggc 2460

accaaggtgg agatcaag 2478

<210> 24

<211> 755

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-OX40 TsAb_M of monomeric form

<400> 24

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

515 520 525

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser

530 535 540

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

545 550 555 560

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

565 570 575

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

Ser Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr Cys Arg Ala

660 665 670

Ser Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu

740 745 750

Ile Lys Arg

755

<210> 25

<211> 2265

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-OX40 TsAb_M of monomeric form

<400> 25

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 agctggtgga gagcggcggc 1560

ggcctggtgc agcccggcgg cagcctgcgc ctgagctgcg ccgccagcgg cttcaccttc 1620

agcagctaca gcatgaactg ggtgcgccag gcccccggca agggcctgga gtgggtgagc 1680

tacatcagca gcagcagcag caccatctac tacgccgaca gcgtgaaggg ccgcttcacc 1740

atcagccgcg acaacgccaa gaacagcctg tacctgcaga tgaacagcct gcgcgacgag 1800

gacaccgccg tgtactactg cgcccgcggc gtgtaccaca acggctggag cttcttcgac 1860

tactggggcc agggcaccct gctgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatccag atgacccaga gccccagcag cctgagcgcc 1980

agcgtgggca accgcgtgac catcacctgc cgcgccagcc aggacatcag cagctggctg 2040

gcctggtacc agcagaagcc cgagaaggcc cccaagagcc tgatctacgc cgccagcagc 2100

ctgcagagcg gcgtgcccag ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 2160

accatcagca gcctgcagcc cgaggacttc gccacctact actgccagca gtacaacagc 2220

taccccctga ccttcggcca gggcacccgc ctggagatca agcgc 2265

<210> 26

<211> 821

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-OX40 TsAb_D of dimeric forms

<400> 26

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

580 585 590

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

595 600 605

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

610 615 620

Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser

625 630 635 640

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

645 650 655

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

660 665 670

Cys Ala Arg Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp

675 680 685

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

690 695 700

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

705 710 715 720

Pro Ser Ser Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr Cys

725 730 735

Arg Ala Ser Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys

740 745 750

Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln

755 760 765

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

770 775 780

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

785 790 795 800

Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg

805 810 815

Leu Glu Ile Lys Arg

820

<210> 27

<211> 2463

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-OX40 TsAb_D of dimeric forms

<400> 27

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

ctggtggaga gcggcggcgg cctggtgcag cccggcggca gcctgcgcct gagctgcgcc 1800

gccagcggct tcaccttcag cagctacagc atgaactggg tgcgccaggc ccccggcaag 1860

ggcctggagt gggtgagcta catcagcagc agcagcagca ccatctacta cgccgacagc 1920

gtgaagggcc gcttcaccat cagccgcgac aacgccaaga acagcctgta cctgcagatg 1980

aacagcctgc gcgacgagga caccgccgtg tactactgcg cccgcggcgt gtaccacaac 2040

ggctggagct tcttcgacta ctggggccag ggcaccctgc tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 2160

cccagcagcc tgagcgccag cgtgggcaac cgcgtgacca tcacctgccg cgccagccag 2220

gacatcagca gctggctggc ctggtaccag cagaagcccg agaaggcccc caagagcctg 2280

atctacgccg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2340

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2400

tgccagcagt acaacagcta ccccctgacc ttcggccagg gcacccgcct ggagatcaag 2460

cgc 2463

<210> 28

<211> 754

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-GITR TsAb_M of monomeric form

<400> 28

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 Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser

515 520 525

Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr

530 535 540

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

545 550 555 560

Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro

565 570 575

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

580 585 590

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

595 600 605

Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln

610 615 620

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

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys

645 650 655

Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala

660 665 670

Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly

675 680 685

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

690 695 700

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

705 710 715 720

Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln

725 730 735

Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

740 745 750

Ile Lys

<210> 29

<211> 2262

<212> DNA

<213> Artificial

<220>

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

<400> 29

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 aggtgaccct gaaggagagc 1560

ggccccggca tcctgaagcc cagccagacc ctgagcctga cctgcagctt cagcggcttc 1620

agcctgagca ccagcggcat gggcgtgggc tggatccgcc agcccagcgg caagggcctg 1680

gagtggctgg cccacatctg gtgggacgac gacaagtact acaaccccag cctgaagagc 1740

cagctgacca tcagcaagga caccagccgc aaccaggtgt tcctgaagat caccagcgtg 1800

gacaccgccg acgccgccac ctactactgc gcccgcaccc gccgctactt ccccttcgcc 1860

tactggggcc agggcaccct ggtgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatcgtg atgacccaga gccagaagtt catgagcacc 1980

agcgtgggcg accgcgtgag cgtgacctgc aaggccagcc agaacgtggg caccaacgtg 2040

gcctggtacc agcagaagcc cggccagagc cccaaggccc tgatctacag cgccagctac 2100

cgctacagcg gcgtgcccga ccgcttcacc ggcagcggca gcggcaccga cttcaccctg 2160

accatcaaca acgtgcacag cgaggacctg gccgagtact tctgccagca gtacaacacc 2220

gaccccctga ccttcggcgc cggcaccaag ctggagatca ag 2262

<210> 30

<211> 820

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-GITR TsAb_D of dimeric forms

<400> 30

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 Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys

580 585 590

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

595 600 605

Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

610 615 620

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr

625 630 635 640

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

645 650 655

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

660 665 670

Thr Tyr Tyr Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp

675 680 685

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

690 695 700

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

705 710 715 720

Gln Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Thr Leu Thr Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe

785 790 795 800

Cys Gln Gln Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys

805 810 815

Leu Glu Ile Lys

820

<210> 31

<211> 2460

<212> DNA

<213> Artificial

<220>

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

<400> 31

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

gtgaccctga aggagagcgg ccccggcatc ctgaagccca gccagaccct gagcctgacc 1800

tgcagcttca gcggcttcag cctgagcacc agcggcatgg gcgtgggctg gatccgccag 1860

cccagcggca agggcctgga gtggctggcc cacatctggt gggacgacga caagtactac 1920

aaccccagcc tgaagagcca gctgaccatc agcaaggaca ccagccgcaa ccaggtgttc 1980

ctgaagatca ccagcgtgga caccgccgac gccgccacct actactgcgc ccgcacccgc 2040

cgctacttcc ccttcgccta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatcgtgat gacccagagc 2160

cagaagttca tgagcaccag cgtgggcgac cgcgtgagcg tgacctgcaa ggccagccag 2220

aacgtgggca ccaacgtggc ctggtaccag cagaagcccg gccagagccc caaggccctg 2280

atctacagcg ccagctaccg ctacagcggc gtgcccgacc gcttcaccgg cagcggcagc 2340

ggcaccgact tcaccctgac catcaacaac gtgcacagcg aggacctggc cgagtacttc 2400

tgccagcagt acaacaccga ccccctgacc ttcggcgccg gcaccaagct ggagatcaag 2460

<210> 32

<211> 752

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-CD40L TsAb_M of monomeric form

<400> 32

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

515 520 525

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

530 535 540

Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu

610 615 620

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

625 630 635 640

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

645 650 655

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

740 745 750

<210> 33

<211> 2256

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-CD40L TsAb_M of monomeric form

<400> 33

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 ggagggagtg aggtgcagct gctggagagc 1560

ggcggcggcc tggtgcagcc cggcggcagc ctgcgcctga gctgcgccgc cagcggcttc 1620

accttcagca gctacgccat gagctgggtg cgccaggccc ccggcaaggg cctggagtgg 1680

gtgagcgcca tcagcggcag cggcggcagc acctactacg ccgacagcgt gaagggccgc 1740

ttcaccatca gccgcgacaa cagcaagaac accctgtacc tgcagatgaa cagcctgcgc 1800

gccgaggaca ccgccgtgta ctactgcgcc aagagctacg gcgccttcga ctactggggc 1860

cagggcaccc tggtgaccgt gagcagcggc ggcggcggca gcggcggcgg cggcagcggc 1920

ggcggcggca gcgacatcca gatgacccag agccccagca gcctgagcgc cagcgtgggc 1980

gaccgcgtga ccatcacctg ccgcgccagc cagagcatca gcagctacct gaactggtac 2040

cagcagaagc ccggcaaggc ccccaagctg ctgatctacg ccgccagcag cctgcagagc 2100

ggcgtgccca gccgcttcag cggcagcggc agcggcaccg acttcaccct gaccatcagc 2160

agcctgcagc ccgaggactt cgccacctac tactgccagc agagctacag cacccccaac 2220

accttcggcc agggcaccaa ggtggagatc aagcgc 2256

<210> 34

<211> 818

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-CD40L TsAb_D of dimeric forms

<400> 34

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 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

580 585 590

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

625 630 635 640

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

645 650 655

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

660 665 670

Tyr Tyr Cys Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Ser Tyr Ser Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

805 810 815

Lys Arg

<210> 35

<211> 2454

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-CD40L TsAb_D of dimeric forms

<400> 35

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 gggcgtggag 1740

gtgcagctgc tggagagcgg cggcggcctg gtgcagcccg gcggcagcct gcgcctgagc 1800

tgcgccgcca gcggcttcac cttcagcagc tacgccatga gctgggtgcg ccaggccccc 1860

ggcaagggcc tggagtgggt gagcgccatc agcggcagcg gcggcagcac ctactacgcc 1920

gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 1980

cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgccaa gagctacggc 2040

gccttcgact actggggcca gggcaccctg gtgaccgtga gcagcggcgg cggcggcagc 2100

ggcggcggcg gcagcggcgg cggcggcagc gacatccaga tgacccagag ccccagcagc 2160

ctgagcgcca gcgtgggcga ccgcgtgacc atcacctgcc gcgccagcca gagcatcagc 2220

agctacctga actggtacca gcagaagccc ggcaaggccc ccaagctgct gatctacgcc 2280

gccagcagcc tgcagagcgg cgtgcccagc cgcttcagcg gcagcggcag cggcaccgac 2340

ttcaccctga ccatcagcag cctgcagccc gaggacttcg ccacctacta ctgccagcag 2400

agctacagca cccccaacac cttcggccag ggcaccaagg tggagatcaa gcgc 2454

<210> 36

<211> 754

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-CD27 TsAb_M of monomeric form

<400> 36

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 Glu Ser Gly Gly Gly Val Val Gln Pro Gly

515 520 525

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

530 535 540

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

545 550 555 560

Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser

565 570 575

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

580 585 590

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

595 600 605

Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Ile Lys

<210> 37

<211> 2262

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the CD19-CD3-CD27 TsAb_M of monomeric form

<400> 37

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 ggtggagagc 1560

ggcggcggcg tggtgcagcc cggccgcagc ctgcgcctga gctgcgccgc cagcggcttc 1620

accttcagca gctacgacat gcactgggtg cgccaggccc ccggcaaggg cctggagtgg 1680

gtggccgtga tctggtacga cggcagcaac aagtactacg ccgacagcgt gaagggccgc 1740

ttcaccatca gccgcgacaa cagcaagaac accctgtacc tgcagatgaa cagcctgcgc 1800

gccgaggaca ccgccgtgta ctactgcgcc cgcggcagcg gcaactgggg cttcttcgac 1860

tactggggcc agggcaccct ggtgaccgtg agcagcggcg gcggcggcag cggcggcggc 1920

ggcagcggcg gcggcggcag cgacatccag atgacccaga gccccagcag cctgagcgcc 1980

agcgtgggcg accgcgtgac catcacctgc cgcgccagcc agggcatcag ccgctggctg 2040

gcctggtacc agcagaagcc cgagaaggcc cccaagagcc tgatctacgc cgccagcagc 2100

ctgcagagcg gcgtgcccag ccgcttcagc ggcagcggca gcggcaccga cttcaccctg 2160

accatcagca gcctgcagcc cgaggacttc gccacctact actgccagca gtacaacacc 2220

tacccccgca ccttcggcca gggcaccaag gtggagatca ag 2262

<210> 38

<211> 820

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the CD19-CD3-CD27 TsAb_D of dimeric forms

<400> 38

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 Glu Ser Gly Gly Gly Val Val Gln

580 585 590

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

595 600 605

Ser Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

610 615 620

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala

625 630 635 640

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

645 650 655

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

660 665 670

Tyr Tyr Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Arg Ala Ser Gln Gly Ile Ser Arg Trp Leu Ala Trp Tyr Gln Gln Lys

740 745 750

Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln

755 760 765

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

770 775 780

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

785 790 795 800

Cys Gln Gln Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys

805 810 815

Val Glu Ile Lys

820

<210> 39

<211> 2460

<212> DNA

<213> Artificial

<220>

<223>The nucleotide acid sequence of the CD19-CD3-CD27 TsAb_D of dimeric forms

<400> 39

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 tggagagcgg cggcggcgtg gtgcagcccg gccgcagcct gcgcctgagc 1800

tgcgccgcca gcggcttcac cttcagcagc tacgacatgc actgggtgcg ccaggccccc 1860

ggcaagggcc tggagtgggt ggccgtgatc tggtacgacg gcagcaacaa gtactacgcc 1920

gacagcgtga agggccgctt caccatcagc cgcgacaaca gcaagaacac cctgtacctg 1980

cagatgaaca gcctgcgcgc cgaggacacc gccgtgtact actgcgcccg cggcagcggc 2040

aactggggct tcttcgacta ctggggccag ggcaccctgg tgaccgtgag cagcggcggc 2100

ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatccagat gacccagagc 2160

cccagcagcc tgagcgccag cgtgggcgac cgcgtgacca tcacctgccg cgccagccag 2220

ggcatcagcc gctggctggc ctggtaccag cagaagcccg agaaggcccc caagagcctg 2280

atctacgccg ccagcagcct gcagagcggc gtgcccagcc gcttcagcgg cagcggcagc 2340

ggcaccgact tcaccctgac catcagcagc ctgcagcccg aggacttcgc cacctactac 2400

tgccagcagt acaacaccta cccccgcacc ttcggccagg gcaccaaggt ggagatcaag 2460

<210> 40

<211> 250

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-CD19 scFv

<400> 40

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> 41

<211> 124

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-CD19 scFv

<400> 41

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> 42

<211> 111

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-CD19 scFv

<400> 42

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> 43

<211> 243

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of AntiCD3 McAb scFv

<400> 43

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 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> 44

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of AntiCD3 McAb scFv

<400> 44

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> 45

<211> 106

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of AntiCD3 McAb scFv

<400> 45

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> 46

<211> 246

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-4-1BB scFv

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

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

130 135 140

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

145 150 155 160

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

165 170 175

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr

180 185 190

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

195 200 205

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

210 215 220

Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr Phe Cys Gly Gly Thr

225 230 235 240

Lys Val Glu Ile Lys Arg

245

<210> 47

<211> 121

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-4-1BB scFv

<400> 47

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 110

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-4-1BB scFv

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

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

100 105 110

<210> 49

<211> 247

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-ICOS scFv

<400> 49

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 Gly Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg 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 Tyr Cys

85 90 95

Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly

115 120 125

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

130 135 140

Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Leu Leu Ala Trp Tyr

165 170 175

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

180 185 190

Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

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

210 215 220

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

225 230 235 240

Gly Thr Lys Val Glu Ile Lys

245

<210> 50

<211> 125

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-ICOS scFv

<400> 50

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 Gly Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro His Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg 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 Tyr Cys

85 90 95

Ala Arg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 107

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-ICOS scFv

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 242

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-OX40 scFv

<400> 52

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

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met

20 25 30

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Ala Ser Val Gly Asn Arg Val Thr Ile Thr Cys Arg Ala Ser

145 150 155 160

Gln Asp Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile

225 230 235 240

Lys Arg

<210> 53

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-OX40 scFv

<400> 53

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

1 5 10 15

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met

20 25 30

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

65 70 75 80

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

85 90 95

Gly Val Tyr His Asn Gly Trp Ser Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Leu Thr Val Ser Ser

115

<210> 54

<211> 108

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-OX40 scFv

<400> 54

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

1 5 10 15

Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 55

<211> 241

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-GITR scFv

<400> 55

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Gln Lys Phe

130 135 140

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

145 150 155 160

Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ser Pro Lys Ala Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val

180 185 190

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

195 200 205

Ile Asn Asn Val His Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln

210 215 220

Tyr Asn Thr Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 56

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-GITR SCFV

<400> 56

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

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

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Arg Arg Tyr Phe Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 57

<211> 107

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-GITR scFv

<400> 57

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 58

<211> 239

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-CD 40 L scFv

<400> 58

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

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

115 120 125

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

130 135 140

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Thr Pro Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

225 230 235

<210> 59

<211> 116

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-CD 40 L scFv

<400> 59

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Lys Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 60

<211> 108

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-CD 40 L scFv

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 61

<211> 241

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of anti-CD27 scFv

<400> 61

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Tyr Asn Thr Tyr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

225 230 235 240

Lys

<210> 62

<211> 119

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the heavy chain variable region of anti-CD27 scFv

<400> 62

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 63

<211> 107

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of the light chain variable region of anti-CD27 scFv

<400> 63

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 750

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CD19 scFv

<400> 64

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> 65

<211> 372

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-CD19 scFv

<400> 65

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> 66

<211> 333

<212> DNA

<213> Artificial

<220>

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

<400> 66

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> 67

<211> 729

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of AntiCD3 McAb scFv

<400> 67

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> 68

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of AntiCD3 McAb scFv

<400> 68

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> 69

<211> 318

<212> DNA

<213> Artificial

<220>

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

<400> 69

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> 70

<211> 738

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-4-1BB scFv

<400> 70

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120

cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180

cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300

cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt gaccgtgagc 360

agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcga gatcgtgctg 420

acccagagcc ccgccaccct gagcctgagc cccggcgagc gcgccaccct gagctgccgc 480

gccagccaga gcgtgagcag ctacctggcc tggtaccagc agaagcccgg ccaggccccc 540

cgcctgctga tctacgacgc cagcaaccgc gccaccggca tccccgcccg cttcagcggc 600

agcggcagcg gcaccgactt caccctgacc atcagcagcc tggagcccga ggacttcgcc 660

gtgtactact gccagcagcg cagcaactgg ccccccgccc tgaccttctg cggcggcacc 720

aaggtggaga tcaagcgc 738

<210> 71

<211> 363

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-4-1BB scFv

<400> 71

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtacggcgg cagcttcagc ggctactact ggagctggat ccgccagagc 120

cccgagaagg gcctggagtg gatcggcgag atcaaccacg gcggctacgt gacctacaac 180

cccagcctgg agagccgcgt gaccatcagc gtggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgcccg cgactacggc 300

cccggcaact acgactggta cttcgacctg tggggccgcg gcaccctggt gaccgtgagc 360

agc 363

<210> 72

<211> 330

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-4-1BB scFv

<400> 72

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 120

ggccaggccc cccgcctgct gatctacgac gccagcaacc gcgccaccgg catccccgcc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag cgcagcaact ggccccccgc cctgaccttc 300

tgcggcggca ccaaggtgga gatcaagcgc 330

<210> 73

<211> 741

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-ICOS scFv

<400> 73

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180

gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300

tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360

gtgaccgtga gcagcggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420

gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 480

atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 540

ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 600

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 660

gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 720

ggcaccaagg tggagatcaa g 741

<210> 74

<211> 375

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-ICOS scFv

<400> 74

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc ggctactaca tgcactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatgggctgg atcaaccccc acagcggcgg caccaactac 180

gcccagaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgcacctac 300

tactacgaca gcagcggcta ctaccacgac gccttcgaca tctggggcca gggcaccatg 360

gtgaccgtga gcagc 375

<210> 75

<211> 321

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-ICOS scFv

<400> 75

gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gggcatcagc cgcctgctgg cctggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgtg gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag gccaacagct tcccctggac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210> 76

<211> 726

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-OX40 scFv

<400> 76

cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60

gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120

aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180

agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240

atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300

aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420

agccccagca gcctgagcgc cagcgtgggc aaccgcgtga ccatcacctg ccgcgccagc 480

caggacatca gcagctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540

ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660

tactgccagc agtacaacag ctaccccctg accttcggcc agggcacccg cctggagatc 720

aagcgc 726

<210> 77

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-OX40 scFv

<400> 77

cagctggtgg agagcggcgg cggcctggtg cagcccggcg gcagcctgcg cctgagctgc 60

gccgccagcg gcttcacctt cagcagctac agcatgaact gggtgcgcca ggcccccggc 120

aagggcctgg agtgggtgag ctacatcagc agcagcagca gcaccatcta ctacgccgac 180

agcgtgaagg gccgcttcac catcagccgc gacaacgcca agaacagcct gtacctgcag 240

atgaacagcc tgcgcgacga ggacaccgcc gtgtactact gcgcccgcgg cgtgtaccac 300

aacggctgga gcttcttcga ctactggggc cagggcaccc tgctgaccgt gagcagc 357

<210> 78

<211> 324

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-OX40 scFv

<400> 78

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcaa ccgcgtgacc 60

atcacctgcc gcgccagcca ggacatcagc agctggctgg cctggtacca gcagaagccc 120

gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacagct accccctgac cttcggccag 300

ggcacccgcc tggagatcaa gcgc 324

<210> 79

<211> 723

<212> DNA

<213> Artificial

<220>

<223>The nucleotide acid sequence of anti-GITR scFv

<400> 79

caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120

cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180

tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240

ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300

cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcgt gatgacccag 420

agccagaagt tcatgagcac cagcgtgggc gaccgcgtga gcgtgacctg caaggccagc 480

cagaacgtgg gcaccaacgt ggcctggtac cagcagaagc ccggccagag ccccaaggcc 540

ctgatctaca gcgccagcta ccgctacagc ggcgtgcccg accgcttcac cggcagcggc 600

agcggcaccg acttcaccct gaccatcaac aacgtgcaca gcgaggacct ggccgagtac 660

ttctgccagc agtacaacac cgaccccctg accttcggcg ccggcaccaa gctggagatc 720

aag 723

<210> 80

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-GITR scFv

<400> 80

caggtgaccc tgaaggagag cggccccggc atcctgaagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc accagcggca tgggcgtggg ctggatccgc 120

cagcccagcg gcaagggcct ggagtggctg gcccacatct ggtgggacga cgacaagtac 180

tacaacccca gcctgaagag ccagctgacc atcagcaagg acaccagccg caaccaggtg 240

ttcctgaaga tcaccagcgt ggacaccgcc gacgccgcca cctactactg cgcccgcacc 300

cgccgctact tccccttcgc ctactggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 81

<211> 321

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-GITR scFv

<400> 81

gacatcgtga tgacccagag ccagaagttc atgagcacca gcgtgggcga ccgcgtgagc 60

gtgacctgca aggccagcca gaacgtgggc accaacgtgg cctggtacca gcagaagccc 120

ggccagagcc ccaaggccct gatctacagc gccagctacc gctacagcgg cgtgcccgac 180

cgcttcaccg gcagcggcag cggcaccgac ttcaccctga ccatcaacaa cgtgcacagc 240

gaggacctgg ccgagtactt ctgccagcag tacaacaccg accccctgac cttcggcgcc 300

ggcaccaagc tggagatcaa g 321

<210> 82

<211> 717

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CD 40 L scFv

<400> 82

gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300

ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagcgg cggcggcggc 360

agcggcggcg gcggcagcgg cggcggcggc agcgacatcc agatgaccca gagccccagc 420

agcctgagcg ccagcgtggg cgaccgcgtg accatcacct gccgcgccag ccagagcatc 480

agcagctacc tgaactggta ccagcagaag cccggcaagg cccccaagct gctgatctac 540

gccgccagca gcctgcagag cggcgtgccc agccgcttca gcggcagcgg cagcggcacc 600

gacttcaccc tgaccatcag cagcctgcag cccgaggact tcgccaccta ctactgccag 660

cagagctaca gcacccccaa caccttcggc cagggcacca aggtggagat caagcgc 717

<210> 83

<211> 348

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-CD 40 L scFv

<400> 83

gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcag cacctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc caagagctac 300

ggcgccttcg actactgggg ccagggcacc ctggtgaccg tgagcagc 348

<210> 84

<211> 324

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-CD 40 L scFv

<400> 84

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gagcatcagc agctacctga actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag agctacagca cccccaacac cttcggccag 300

ggcaccaagg tggagatcaa gcgc 324

<210> 85

<211> 723

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of anti-CD27 scFv

<400> 85

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300

ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagcggc 360

ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcgacatcca gatgacccag 420

agccccagca gcctgagcgc cagcgtgggc gaccgcgtga ccatcacctg ccgcgccagc 480

cagggcatca gccgctggct ggcctggtac cagcagaagc ccgagaaggc ccccaagagc 540

ctgatctacg ccgccagcag cctgcagagc ggcgtgccca gccgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaccatcagc agcctgcagc ccgaggactt cgccacctac 660

tactgccagc agtacaacac ctacccccgc accttcggcc agggcaccaa ggtggagatc 720

aag 723

<210> 86

<211> 357

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the heavy chain variable region of anti-CD27 scFv

<400> 86

caggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agctacgaca tgcactgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtggccgtg atctggtacg acggcagcaa caagtactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcggcagc 300

ggcaactggg gcttcttcga ctactggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 87

<211> 321

<212> DNA

<213> Artificial

<220>

<223>The nucleotide sequence of the light chain variable region of anti-CD27 scFv

<400> 87

gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga ccgcgtgacc 60

atcacctgcc gcgccagcca gggcatcagc cgctggctgg cctggtacca gcagaagccc 120

gagaaggccc ccaagagcct gatctacgcc gccagcagcc tgcagagcgg cgtgcccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacacct acccccgcac cttcggccag 300

ggcaccaagg tggagatcaa g 321

<210> 88

<211> 19

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence of secreting, expressing signal peptide

<400> 88

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

1 5 10 15

Ser Arg Ala

<210> 89

<211> 57

<212> DNA

<213> Artificial

<220>

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

<400> 89

atgacccggc tgaccgtgct ggccctgctg gccggcctgc tggcctcctc cagggcc 57

<210> 90

<211> 59

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-Sig-F

<400> 90

gtgctggata tctgcagaat tcgccgccac catgacccgg ctgaccgtgc tggccctgc 59

<210> 91

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Sig-R

<400> 91

ggccctggag gaggccagca ggccggccag cagggccagc acggtcagc 49

<210> 92

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Sig-CD19-F

<400> 92

ctgctggcct cctccagggc cgacatccag ctgacccaga gc 42

<210> 93

<211> 23

<212> DNA

<213> Artificial

<220>

<223> CD19-R

<400> 93

gctgctcacg gtcacggtgg tgc 23

<210> 94

<211> 56

<212> DNA

<213> Artificial

<220>

<223> CD19-G4S-CD3-F

<400> 94

ccaccgtgac cgtgagcagc ggtggcggag ggtccgacat caagctgcag cagagc 56

<210> 95

<211> 20

<212> DNA

<213> Artificial

<220>

<223> CD3-R

<400> 95

cttcagctcc agcttggtgc 20

<210> 96

<211> 72

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-4-1BB-F

<400> 96

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gc 72

<210> 97

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-4-1BB-R

<400> 97

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 98

<211> 41

<212> DNA

<213> Artificial

<220>

<223> CD3-IgD-F

<400> 98

gcaccaagct ggagctgaag gccagcaaga gcaagaagga g 41

<210> 99

<211> 21

<212> DNA

<213> Artificial

<220>

<223> IgD-R

<400> 99

cacgcccagg ggctgggtgt g 21

<210> 100

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-4-1BB-F

<400> 100

cacacccagc ccctgggcgt gcaggtgcag ctgcagcagt gg 42

<210> 101

<211> 86

<212> DNA

<213> Artificial

<220>

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

<400> 101

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gcagctggtg cagagc 86

<210> 102

<211> 50

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-ICOS-R

<400> 102

ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc 50

<210> 103

<211> 42

<212> DNA

<213> Artificial

<220>

<223> IgD-ICOS-F

<400> 103

cacacccagc ccctgggcgt gcaggtgcag ctggtgcaga gc 42

<210> 104

<211> 85

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-OX40-F

<400> 104

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccagct ggtggagagc ggcgg 85

<210> 105

<211> 52

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-OX40-R

<400> 105

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaggcgggt gc 52

<210> 106

<211> 45

<212> DNA

<213> Artificial

<220>

<223> IgD-OX40-F

<400> 106

gccacaccca gcccctgggc gtgcagctgg tggagagcgg cggcg 45

<210> 107

<211> 85

<212> DNA

<213> Artificial

<220>

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

<400> 107

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gaccctgaag gagag 85

<210> 108

<211> 52

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-GITR-R

<400> 108

ctgatcagcg gtttaaactt aagctttcac ttgatctcca gcttggtgcc gg 52

<210> 109

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-GITR-F

<400> 109

gccacaccca gcccctgggc gtgcaggtga ccctgaagga gag 43

<210> 110

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD40L-F

<400> 110

ggcaccaagc tggagctgaa gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 60

ggcagcgagg tgcagctgct ggagagc 87

<210> 111

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD40L-R

<400> 111

ctgatcagcg gtttaaactt aagctttcag cgcttgatct ccaccttggt g 51

<210> 112

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-CD40L-F

<400> 112

gccacaccca gcccctgggc gtggaggtgc agctgctgga gag 43

<210> 113

<211> 86

<212> DNA

<213> Artificial

<220>

<223> CD3-(GGGGS)3-CD27-F

<400> 113

gcaccaagct ggagctgaag ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg 60

gcagccaggt gcagctggtg gagagc 86

<210> 114

<211> 51

<212> DNA

<213> Artificial

<220>

<223> pcDNA3.1-CD27-R

<400> 114

ctgatcagcg gtttaaactt aagctttcac ttgatctcca ccttggtgcc c 51

<210> 115

<211> 43

<212> DNA

<213> Artificial

<220>

<223> IgD-CD27-F

<400> 115

gccacaccca gcccctgggc gtgcaggtgc agctggtgga gag 43

Claims (16)

1. a kind of three functional moleculars, structure include that the first functional domain of CD19 can be incorporated into, can combine and activate T thinSecond functional domain of cellular surface CD3 molecules and the third functional domain that can combine and activate the positive costimulatory molecules of T cell.

2. three functional molecular according to claim 1, which is characterized in that three functional molecular can be identification CD19'sMeanwhile with reference to and activate T cell surface C D3 molecules and the positive costimulatory molecules of T cell, so as to generate needed for T cell activationOne signal and second signal.

3. three functional molecular according to claim 1, which is characterized in that first functional domain is the antibody of anti-CD19,Second functional domain is the antibody of AntiCD3 McAb, and the third functional domain is the antibody of the positive costimulatory molecules of anti-T cell.

4. three functional molecular according to claim 3, which is characterized in that the antibody is selected from Fab antibody, Fv antibody or listChain antibody.

5. three functional molecular according to claim 1, which is characterized in that first functional domain and second functional domainBetween connected by junction fragment 1, connected between second functional domain and the third functional domain by junction fragment 2.

6. three functional molecular according to claim 5, which is characterized in that the junction fragment 1 and junction fragment 2 be selected fromG4S is the junction fragment of unit or the hinge area segment of Immunoglobulin IgD.

7. three functional molecular according to claim 6, which is characterized in that the amino acid of the junction fragment as unit of G4SSequence such as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 it is any shown in；The hinge area segment of Immunoglobulin IgDAmino acid sequence as shown in SEQ ID NO.7.

8. three functional molecular according to claim 1, which is characterized in that first functional domain is the single-stranded anti-of anti-CD19Body, second functional domain are the single-chain antibody of AntiCD3 McAb, and the third functional domain is single-stranded for the positive costimulatory molecules of anti-T cellAntibody, the single-chain antibody include heavy chain variable region and light chain variable region.

9. three functional molecular according to claim 8, which is characterized in that the weight chain variable of the single-chain antibody of the anti-CD19The amino acid sequence in area is as shown in SEQ ID NO.41；The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-CD19As shown in SEQ ID NO.42；The amino acid sequence of the heavy chain variable region of the single-chain antibody of the AntiCD3 McAb such as SEQ ID NO.44It is shown；The amino acid sequence of the light chain variable region of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.45；The anti-T is thinThe single-chain antibody of the positive costimulatory molecules of born of the same parents is selected from the single-chain antibody of anti-4-1BB, the single-chain antibody of anti-ICOS, the single-stranded of anti-OX40 and resistsBody, the single-chain antibody of anti-GITR, the single-chain antibody of anti-CD 40 L or anti-CD27 single-chain antibody it is any；The list of the anti-4-1BBThe amino acid sequence of the heavy chain variable region of chain antibody is as shown in SEQ ID NO.47；The light chain of the single-chain antibody of the anti-4-1BBThe amino acid sequence of variable region is as shown in SEQ ID NO.48；The amino acid of the heavy chain variable region of the single-chain antibody of the anti-ICOSSequence is as shown in SEQ ID NO.50；The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-ICOS such as SEQ IDShown in NO.51；The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-OX40 is as shown in SEQ ID NO.53；It is describedThe amino acid sequence of the light chain variable region of the single-chain antibody of anti-OX40 is as shown in SEQ ID NO.54；The anti-GITR's is single-stranded anti-The amino acid sequence of the heavy chain variable region of body is as shown in SEQ ID NO.56；The light chain variable region of the single-chain antibody of the anti-GITRAmino acid sequence as shown in SEQ ID NO.57；The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD 40 LAs shown in SEQ ID NO.59；The amino acid sequence of the light chain variable region of the single-chain antibody of the anti-CD 40 L such as SEQ IDShown in NO.60；The amino acid sequence of the heavy chain variable region of the single-chain antibody of the anti-CD27 is as shown in SEQ ID NO.62；It is describedThe amino acid sequence of the light chain variable region of the single-chain antibody of anti-CD27 is as shown in SEQ ID NO.63.

10. three functional molecular according to claim 9, which is characterized in that the amino acid of the single-chain antibody of the anti-CD19Sequence is as shown in SEQ ID NO.40；The amino acid sequence of the single-chain antibody of the AntiCD3 McAb is as shown in SEQ ID NO.43；It is describedThe amino acid sequence of the single-chain antibody of anti-4-1BB is as shown in SEQ ID NO.46；The amino acid of the single-chain antibody of the anti-ICOSSequence is as shown in SEQ ID NO.49；The amino acid sequence of the single-chain antibody of the anti-OX40 is as shown in SEQ ID NO.52；InstituteThe amino acid sequence of the single-chain antibody of anti-GITR is stated as shown in SEQ ID NO.55；The amino of the single-chain antibody of the anti-CD 40 LAcid sequence is as shown in SEQ ID NO.58；The amino acid sequence of the single-chain antibody of the anti-CD27 is as shown in SEQ ID NO.61.

11. three functional molecular according to claim 1, which is characterized in that the amino acid sequence of three functional molecular is such asSEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.22、SEQ ID NO.24、SEQ IDNO.26, SEQ ID NO.28, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.34, SEQ ID NO.36 or SEQID NO.38's is any shown.

12. a kind of polynucleotides, coding three functional moleculars as described in any one of claim 1~11.

13. a kind of expression vector contains polynucleotides as claimed in claim 12.

14. a kind of host cell is converted by expression vector as claimed in claim 13.

15. the preparation method of three functional moleculars as described in any one of claim 1~11, including：Structure contains three functional molecularsThen expression vector containing three functional molecular gene orders is converted into host cell induction table by the expression vector of gene orderIt reaches, is detached from expression product and obtain three functional moleculars.

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