CN1894413A - Activation of tumor reactive lynphatic cell of identifying CD3 or 4-1BB antibody or gene mediation - Google Patents

Abstract

An improved method for ex-vivo generation of tumor-reactive T lymphocyte is provided, comprising, for example, culturing pbmc from a patient with cancer with autologous tumor cells and magnetic beads that can activate t lymphocytes via cd3 in combination with cd28, cd40, or cd28 plus cd40. The invention also provides genes encoding anti-cd3 or anti-41bb scfv and methods to transfect cells of neoplastic or normal origin for expression of those genes at their surface. The genes and transfected tumor cells are useful compositions for induction of a tumor-destructive immune response.

Description

Identification CD3 or the antibody of 4-1BB or the tumor response lymphocyte activation of gene mediated

The cross reference of related application

The Application No. that present patent application requires on March 26th, 2002 to submit is 10/107,991 right of priority.

With the relevant statement of federal government's sponsored research

This part of work is by the U.S.'s state-run commune hospital patronage, and United States Government can enjoy certain rights and interests in this invention.

Invention field

The present invention relates to produce the lymphocytic method of tumor response T, tumor vaccine and compositions related prepares this class vaccine and compositions related method thereof, and uses these vaccines and compositions related method thereof, and for example interior therapeutic is used.

Background of invention

The following description school bag has been drawn together understanding current this invention Useful Information.This is not to admit that any information provided herein is prior art, and is perhaps relevant with the invention of current description or requirement, and any publication perhaps specific reference or that quote unintentionally or file are prior art.

Human tumor can be expressed kinds of tumors antigen, and wherein most of antigens also are present in some normal tissues, although its expression level very low (Hellstrom and Hellstrom, AdvCancer Res, 12,167-223,1969; Cheever et al., Immunol Rev, 145,33-59,1995; Finn et al., Immunol Rev, 145,61-89,1995; Boon et al., ImmunolToday, 18,267-8,1997; Hellstrom and Hellstrom, Handbook ofExperimental Pharmacology, Vaccines (Chapter 17), 463-478,1999).Many these antigens in suffering from the tumour patient body be have immunogenic.For example, the SEREX technology can detect IgG antibody (Old and Chen, the J.Exp.Med. of kinds of tumors related antigen, 187,1163-1167,1998), by using the tetramer method can confirm T cell recognition tumour antigen (Cassian et al., J.Immunol., 162,1999), by confirming this identification (Boon, Coulie et al. equally at external generation tumor-selective T cell clone, Immunol Today, 18,267-8,1997).This just provides the chance of carrying out the panimmunity treatment for the patient, comprise the immune T lymphocyte (Rosenberg that uses at amplification in vitro to the patient, Biologic Therapy of Cancer (Chapter 19), 487,1995) and curative tumor vaccine (Nestle et al., Nat Med, 4,328-32,1998; Rosenberget al., Nat Med, 4,321-7,1998; Greenberg, Adv Immunol, 49,281-355,1991; Melief and Kast, Immunol Rev, 145,167-77,1995; Pardoll, Curr Opin Immunol, 8,619-21,1996; Hellstrom and Hellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478,1999).

Mouse tumor provides practical disease model for the effective more immunotherapy of exploitation.The reaction object of the destructive immunne response of they meeting expressing tumors is although this need obtain to resist effective immunne response of most spontaneous tumors by specific experimental implementation.(Greenberg，Adv?Immunol，49，281-355，1991；Ken-and?Mule，J.Leuko.Biol.，56，210-214，1994；Cheever，Disis?et?al.，Immunol?Rev，145，33-59，1995；Finn，Jerome?et?al.，Immunol?Rev，145，61-89，1995；Meliefand?Kast，Immunol?Rev，145，167-77，1995；Pardoll，Cun-Opin?Immunol，8，619-21，1.996；Boon，Coulie?et?al.，Immunol?Today，18，267-8，1997；Hellstrom?and?Hellstrom，Handbook?of?Experimental?Pharmacology，Vaccines(Chapter?17)，463-478，1999)。

T lymphocyte (CD8 ⁺And CD4 ⁺) producing the effect of bringing into play key in (also finishing usually) process in the immunne response of tumor destruction, natural killer cell and antibody, scavenger cell have participated in this process (Hellstrom and Hellstrom too, Adv CancerRes, 12,167-223,1969; Greenberg, Adv Immunol, 49,281-355,1991; Meliefand Kast.Immunol Rev, 145,167-77,1995; Hellstrom andHellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478,1999).The antigen presentation of the dendritic cells (DC) that is differentiated by hemopoietic stem cell in the marrow and the monocyte in the blood is normal (Huangct al..Science, 264,961-5,1994), under specific situation, this antigen presentation also can be finished (Chen et al..Cell, 71 by tumour cell self, 1093-102,1992; Schoenberger et al..Cancer Res, 58,3094-100,1998).Promote that the antigenic operation steps of dendritic cells (DC) expressing tumor is most important to obtaining effective tumour immunity, nearest data show, this immunity make to specific human tumor carry out more effective treatment may become (referring to, for example, (Kugler et al..Nature Medicine, 6,332-, 2000)).The active CD8 of vitro cytotoxicity T lymphocyte (CTL) is arranged ⁺The T lymphocyte with combining of the helper T cell that generates lymphokine, all is necessary (Hellstrom and Hellstrom to the removing of most tumors, Handbook of ExperimentalPharmacology, Vaccines (Chapter 17), 463-478,1999).Although it all is favourable (Rodolfo et al., T Immunol, 163,1923-8 that Th1 and Th2 reply, 1999), but Th1 replys and is still playing the part of main role (Hu etal., J Immunol aspect the immune clearance of tumour, 161,3033-41,1998).

Want induce immune response, will carry out common stimulation, particularly by CD80 and/or CD86 on the antigen presenting cell (APC), and the interaction between the CD28 on the T lymphocyte, be this process necessary (Schwartz, the Cell of inducing, 57,1073-81,1989; June et al., Immunol Today, 11.211-6,1990; Linsley and Ledbetter, Annu Rev Immunol, 11,191-212,1993).This causes interleukin-2, interferon-and other promote the lymphokine of immunne response to continue to generate (Thompson et al., Proc NatiAcad Sci USA, 86,1333-7,1989), gene transfection tumour cell with the coding lymphokine can reach above-mentioned identical purpose (Pardoll, Curr Opin Immunol, 8,619-21,1996).The second signal that produces by CD28 even if TXi Baoshouti (TCR) contacts with antigen, can not bring out immunne response yet, even can cause reactionless.Most tumors is not expressed CD80 or CD86 (Chen, Ashe et al., Cell, 71,1093-102,1992; Yang et al., J Immunol, 154,2794-800,1995), reach the lymphoglandula of metastases at antigen, and expressed before dendritic cells (DC) identification, processing and the submission of CD80 and CD86, can not bring out efficient immune (Huang, Golumbek et al..Science, 264,961-5,1994; Yang et al., J Immunol, 158,851-8,1997).This just can be interpreted as what tumour before growing up to agglomerate usually in " under situation about not discovered by " immune monitoring.CD80 and CD86 can not only combine with CD28, and the avidity between the CTLA4 on itself and the T cell that is activated is higher.This combination of back can be brought out a kind of negativity signal (Thompson, Lindsten et al., ProcNati Acad Sci USA, 86,1333-7,1989 that can end immunne response; Walunas et al., Immunity, 1,405-13,1994; Krummel and Allison, J Exp Med, 183,2533-40,1996; Leach et al..Science, 271,1734-6,1996; Walunas et al., J Exp Med, 183,2541-50,1996; Allison et al., Novartis Found Symp, 215,92-8,1998), this illustrate its with CD28 combined the treatment meaning, do not have with combining then of CTLA4.

Although immunity system has the potential of inducing antitumor immunity, it still is a relative nullity to eliminating the tumour formed.By conventional tumor vaccine, or, be difficult to destroy millions of tumour cells by in immune t-cell inductive immunne response vitro conversion, that have anti-tumor activity.Confirmed that this phenomenon is relevant with some escape mechanisms, these escape mechanisms can protect tumour cell to avoid immune attack (Hellstrom and Hellstrom, Handbookof Experimental Pharmacology, Vaccines (Chapter 17), 463-478,1999; Kiessling et al..Cancer Immunol.Immunother., 48,353-362,1999).These mechanism comprise tumour antigen determinant (Maeurer et al., J Clin Invest, 98,1633-41,1996) and/or these antigen presentations can be given I class major histocompatibility antigen (MHC) molecule (Restifo, 1993 of tumor-killing T lymphocyte (CTL); Maeurer, 1996; Hellstrom, 1997; Garrido, 1997; Johnsen, 1998) the lymphocytic elimination of tumor response (Hahne et al., Science, 274,1363-1366,1996 of losing and realizing by apoptosis; Shirald et al., Proc Nati Acad Sci USA, 94,6420-5,1997; Bennett et al., J Immunol, 160,5669-75,1998; Kume etal., Int J Cancer, 84.339-43,1999) (Chappell and Restifo, CancerImmunol Immunother, 47,65-71,1998).Yet, can express immunogenic cancer antigen, and the tumour of induced reaction lymphocyte generation apoptosis not, can under the immunity monitoring, escape usually.This gives the credit to various " BFs " that produced by tumour and/or host cell, and directly, perhaps the antigen presenting cell (APC) by having or not having antigenic determinant to select acts on the T cell indirectly and plays a role.These BFs comprise the tumour antigen and the immunocomplex of solubility, and transforming growth factor-beta (TGF-β), prostaglandin(PG), nitrogen protoxide (NO) or the like (Hellstrom and Hellstrom, Adv Immunol, 18,209-77,1974; Kehrl et al..J Exp Med, 163,1037-50,1986; Sulitzeanu, AdvCancer Res, 60,247-267,1993; Kiessling et al..Springer Semin.Immunopathol., 18,227-242,1996; Kiessling, Wasserman et al..CancerImmunol.Immunother., 48,353-362,1999).Can destroy Th1 reaction (the Hu.Urba et al. of tumour from generation, J Immunol, 161,3033-41,1998) to Th2 reaction (Fiorentino et al. takes place, J Exp Med, 170,2081-95,1989) in the transforming process, the antigen that is discharged by tumour cell forms the downward modulation of immunne response that immunocomplex causes separately or with antibody, and may supervene TGF-β, produces for example such Th2 lymphokine (the Wilbanks et al. of IL-10 then, Eur J Immunol, 22,165-73,1992; D ' Orazioand Niederkom, J Immunol, 160,2089-98,1998).Interaction between CTLA4 on the T lymphocyte that is activated and the CD80/CD86 on the APC or the T cell that is activated also can cause this downward modulation (Leach, Krummel et al., Science.271,1734-6,1996; Allison, Chambers et al., Novartis Found Symp, 215,92-8,1998), TGF-β also can mediate this downward modulation (Chen et al., J Exp Med, 188,1849-57,1998).Confirmed that scavenger cell institute's role in the downward modulation process is to generate nitrogen protoxide (NO) and prostaglandin(PG) (Kiessling, Wasserman et al..CancerImmunol.Immunother., 48,353-362,1999).

Point out in many reports that in tumor-infiltrated T lymphocyte, T cell signaling mechanism usually defective can take place, this has reacted t cell responses and has been suppressed (Mizoguchi etal., Science, 258,1795-8,1992; Nakagomi et al..Cancer Res, 53,5610-5612,1993; Kiessling, Wasserman et al..Cancer Immunol.Immunothcr., 48,353-362,1999), lymphocyte is taken out this inhibition in back in the body can recover.

Here enumerated the diagram of situation when some very big tumours are repelled by immunity system, for example, about using report (Melero etal., Nat Med, 3,682-5,1997 of T cell activation molecule 4-1BB treatment mouse tumor; Melero et al., Eur J Immunol, 28,1116-21,1998).But, more surprising example may be to the early stage observation of renal transplantation, in some accept the renal transplant recipients body, the big metastasis of coming (the Wilson et al. that after removing immunosuppression, has been completely recovered by the tumour cell development of taking from the corpse kidney, N Engi J Med, 278,479-83,1968; Matter et al.Transplantation, 9,71-4,1970).

Cell surface molecule 4-1BB expresses (DeBenedette et al., J Exp Med, 181,985-92,1995 at activatory rather than on natural T cell; Shuford et al., J ExpMed, 186,47-55,1997).Participating in of 4-1BB can be amplified derivative immunne response.Having can stimulate after reporting 4-1BB and the monoclonal antibody of anti-4-1BB contacting by CD8 antigen activates, that the tumour-specific killing activity is arranged ⁺The T lymphopoiesis, stimulate the cytokine (interleukin-2 (IL-2) of interferon-(IFN-γ) and other Th1 types, tumour necrosis factor (TNF-α)) generation/release, and protection (the Hurtadoet al. of the apoptotic T cell of stimulation antagonism, J Immunol, 158,2600-9,1997; Kirn et al., Eur JImmunol, 28,881-90,1998; Natoli et al., Biochem Pharmacol, 56,915-20,1998; Takahashi et al., J Immunol, 162,5037-40,1999; Tsushima et al., Exp Hematol, 27,433-40,1999).In addition, 4-1BB has immunomodulatory effect, comprises the adjusting (Melero et al..Cell Immunol, 190,67-72,1998) to NK 1.1 cells.

Also there is report to point out, the monoclonal antibody of 4-1BB has in the antagonism mouse body, the activity that comprises the tumour that has grown up to (about 10 millimeters of diameter) of the tumour of reduced immunogenicity has generated the CD8 that dissolved cell activity increases with the lymphocyte of the mouse of anti-4-1BB mab treatment ⁺Tumour-specific killer T cell (Melero, Shuford et al., NatMed, 3,682-5,1997).Lymphocyte contacts with the tumour cell of transfected integration 4-1BB part (4-1BBL), and this part combines with 4-1BB, also can significantly amplify CD8 ⁺T is lymphocytic to be replied, and the tumour cell of 4-1BBL transfection is used as therapeutic activity when vaccine is used for mouse model.Yet the curative effect for the treatment of with the monoclonal antibody of anti-4-1BB obviously is better than making of the tumour cell of expressing 4-1BBL the effect of vaccine therapy.Antybody therapy is very effective (Melero for the sarcoma Ag104 of treatment non-immunogenic, Shuford etal., Nat Med, 3,682-5,1997), with the Ag104 cell transfecting and express 4-1BBL, combine with the cell of expressing CD80 again, just can obtain to resist the curative effect (Melero, 1998) of this tumour.

The method that a kind of selectable increase tumour immunity is arranged, be exactly to use once anti--CD3 monoclonal antibody to the patient, can make polyclone T cell activation like this, comprise and activate any tumor response T lymphocyte, be reported in some researchs of carrying out with mouse model, have some to adopt successful experience (the Ellenhom et al. of this method treatment, Science, 242,569-71,1988).

Tumor response T lymphocyte can be used in the body after external generation, the transfer of the tumour immunity that realizes by lymphocyte, and prevention is from transplanted cells hypertrophy (the Klein et al. of each tumor neogenetic thing, Cancer Res, 20,1561-1572,1960).Once relevant in the past after immune lymphocyte adoptive transfer, repel little, form the report (Hellstrom et al., Transplant Proc, 1,90-4,1969) of tumour.The lymphocyte of adoptive transfer can preferentially be positioned at (Mule around the tumour ^*Et al., J.Immunol., 123,600-606,1979), the tumor infiltrating lymphocyte (TIL) that is endowed immunity is used for the treatment of this invention has enlarged its external range of application (Rosenberg, Biologic Therapy ofCancer (Chapter 19), 487,1995).Although seen breathtaking clinical response on one's body in small number of patients, surpass several millimeters tumour at treatment mouse intravital diameter, and when the people treated, successful degree not ideal enough usually (Greenberg, AdvImmunol, 49,281-355,1991; Chen, Ashe et al..Cell.71,1093-102,1992; Meliefand Kast, Immunol Rev, 145,167-77,1995; Hellstrom andHellstrom, Handbook of Experimental Pharmacology, Vaccines (Chapter 17), 463-478,1999).This failure is owing to the previous lymphocyte location of mistake that " escape " mechanism and/or immersion tumour entity are discussed causes.

Therefore need carry out comprising the method that structure can be induced the tumor vaccine of strong immune response more and be generated the T lymphocyte that is used for the treatment of tumour patient.

Brief summary of the invention

The present invention relates to the lymphocytic method of produced in vitro tumor response T, in vivo the tumor vaccine of therapeutic application and relevant composition.

A kind of method is to take from the monokaryon lymphocyte of peripheral blood or tumour, for example takes from the monokaryon lymphocyte of tumour patient, in the presence of specific C D3 and CD28 immobilization antibody with the autologous tumor cell co-cultivation.Just can finish such cultivation through a cycle of 4-5 days.Can be with cell amplification to the level that can be used in treatment, for example after removal is attached with the magnetic bead of immobilization antibody in the interleukin-2 of 10 μ l/ml.These methods are of great use to the lymphocytic production of tumor response that promotion is used for the treatment of tumour.Under not by the situation of any special theory or mechanism constraint, the operation of this invention can be interpreted as following process: the T lymphocyte of expressing low-level CD3 is activated by polyclone, and hyperplasia is vigorous, produces Th1 type lymphokine, and destroy tumour cell, release tumor antigen rapidly.Can also think that it is dendritic cell that the lymphocytic activation of polyclone T makes the monocyte maturation in the substratum, it can engulf dead tumour cell, processes and offer tumour antigen, and inducing tumor-specific T cell comprises the lasting amplification of CTL.

The present invention also provides, and for example, is coded in the gene of the anti-CD3 of tumor cell surface expression or anti-4-1BB strand Fv (scFv) molecule and with the cell of these gene transfections, is used for treatment for cancer in the body.Under not by the situation of any special theory or mechanism constraint, can think and induce polyclone T lymphocyte activator at the anti--CD3 scFv of tumor cell surface expression, and destroyed tumour cell, release tumor antigen, promote the conversion of the tumour immunity of antigen-specific, promptly we can detect the repulsion of identical tumour to " wild-type " (non-transfection) cell.Also can think at the anti-4-1BB scFv molecule of the expression of tumor cell surface by constantly making the amplification of tumor response T lymphocyte and/or by protecting them that apoptosis does not take place; thereby generate the tumor response lymphokine; interferon-for example, the lymphocytic activation of the reactive T of induced tumor/amplification.With transfected, can be after the tumour cell immunity of the anti-4-1BB scFv of cell surface expression molecule, under without any special theoretical or mechanism constraint, can think that the wild-type cell from same tumour can be by comprising natural killer cell and CD4 ⁺Cell-stimulating is ostracised in interior mechanism.Tumour cell at the anti-4-1BB scFv of cell surface expression molecule is activated when treating the wild-type tumour that has grown up to.

The present invention makes the method for two kinds of new treatment tumours become possibility.At first, its can by for example be attached with anti--CD3/ anti--the CD28/ anti-CD 40 (or anti--CD3/CD28) magnetic bead of immobilization antibody, make " repressed " lymphocyte activator, and make their hyperplasia, generate the Th1 lymphokine, become simultaneously TGF-β is suppressed more insensitive.Under without any special theoretical or mechanism constraint, can think that activated lymphocytes can destroy tumour cell, thereby tumour antigen is provided, and the maturation of inducing antigen presenting cell.Can think that this method can make tumor response CD8 ⁺And CD4 ⁺T lymphocyte and natural killer cell increase within a certain period of time, and this has just adapted to the needs of their adoptive transfers being given tumour patient better.Secondly, this method can provide gene or polynucleotide, and it is for example encoded, the anti--CD3 or the anti-4-1BBscFv of tumor surface, and these molecular energies are effectively induced the tumor destruction immunne response of antagonism from the wild-type cell of same tumour.

Other aspects of this invention are set forth in following claim, complete herein quoting as a reference.

The accompanying drawing summary

Although having wide range of applications of this invention is not limited to here the embodiment that describes or mention of institute, can more understanding be arranged to some aspects of inventing with reference to accompanying drawing.

Fig. 1. take from the in-vitro multiplication of advanced ovarian cancer (OV44) patient's tumor infiltrating lymphocyte (TILs).A has shown that adding autologous tumor cell with the contrast magnetic bead stimulates lymphocytic amplification.B has shown that adding autologous tumor cell with anti--CD3/CD28/CD40 coupling magnetic bead stimulates the lymphocytic amplification in back jointly.C has shown with the anti--CD3/CD28/CD40 magnetic bead that does not add tumour cell stimulates the lymphocytic amplification in back.

Fig. 2. from body, but not allochthonous tumour cell and take from colorectal carcinoma peripheral blood of patients monocyte propagation by the magnetic bead zygotic induction of CD3 and CD28 combination of stimulation.This figure has shown and is having under the situation of the ovarian cancer cell of body (1C) or allogeneic (4007) that stimulated in vitro was taken from the amplification of tumour patient peripheral blood lymphocyte after five days.Used stimulator is: A=contrasts magnetic bead; B=resists-CD28/CD40 coupling magnetic bead; C=resists-CD3/CD28 coupling magnetic bead; D=resists-CD3/CD28/CD40 coupling magnetic bead; E=resists-CD3/CD40 coupling magnetic bead.

Fig. 3. take from and suffer from colorectal carcinoma peripheral blood of patients monocyte under the situation that magnetic bead exists, at 4 hours ⁵¹Show the tumour cell of its cleavable from body during Cr discharges and analyzes, described magnetic bead stimulates CD3 and CD28, or the combination of CD3 and CD28 and CD40.This figure has shown the cell-mediated cytotoxicity that the peripheral blood lymphocyte of taking from patient 1C shows, added anti--CD3/CD28 magnetic bead (A) or quilt by tumour cell after the tumour cell of body adds anti--CD3/CD28/CD40 magnetic bead (B) activation at peripheral blood lymphocyte, on the target cell that is labeled, detect from body.

Fig. 4. take from and suffer from tumor of head and neck peripheral blood of patients monocyte, with autologous tumor cell with can the generation interferon-after stimulating the magnetic bead co-cultivation of CD3 and CD28.This figure has shown that taking from late period tumor of head and neck peripheral blood of patients lymphocyte generates interferon-.The level of interferon-is as shown by using separately anti--CD3/CD28 (A) in the culture supernatant, and anti--CD3/CD28 magnetic bead adds autologous tumor cell (B), or after the contrast magnetic bead adds autologous tumor cell (C) and stimulate, and measures at different time to draw.

Fig. 5 .CD83 is after the CD3 that stimulates with magnetic bead and CD28 stimulation, expresses on the colorectal carcinoma peripheral blood of patients monocyte taking from.This figure has shown the expression of taking from CD83 on late tumor (38C) the patient peripheral blood lymphocytes.With cell with anti--CD83 antibody before stimulating (0 day) directly with phycoerythrin (PE) coupling, or after stimulating 1 day with resisting-coupling of CD3/CD28 magnetic bead.

Fig. 6. by CD3 and the common expressed two days later CD83 level of colorectal carcinoma peripheral blood of patients monocyte of taking from that stimulates of CD28, stimulate the expression level height of back CD83 jointly than add CD40 again with CD3 and CD28.This figure has shown and has taken from late tumor (38C) peripheral blood of patients monocyte that these cells are not activated, or stimulate two days with anti--CD3/CD28/CD40 magnetic bead, or stimulate two days with anti--CD3/CD28 magnetic bead as shown.With cell use simultaneously with anti--CD3 link coupled fluorescence dye and with anti--CD83 link coupled phycoerythrin dyeing.

Fig. 7. compare to some extent with K1735 cell of expressing mouse CD80 and wild-type K1735 cell and disappear through the K1735 melanoma cell that transfection can-CD3scFv (500A2) anti-at cell surface expression.

Fig. 8. be transplanted to the disappearing of K-1735 wild-type cell of homology mouse, described mouse repeatedly immunity with opposing K1735-500A2 cell.With K1735M2/500A2 (2 * 10 ⁶/ mouse) cell carries out three immunity to the C3H/HeN mouse, and each immunity seven days at interval gives mouse with 1 * 10 again ⁶The concentration inoculation K1735M2/500A2 wild-type cell an of/mouse (blue line).With PBS the C3H/HeN mouse is carried out immunity, the K1735M2 with same dose gives mouse inoculation (red line) then.

Fig. 9. Anti-Human CD3scFv gene order, i.e. the sequence of Anti-Human CD3scFv-hIgGl-CD80TM synthetic polyribonucleotides, a kind of product at cell surface expression of encoding can be used in transfection.

Figure 10. after the reverse transcription virus gene transduction, Anti-Human CD3 scFv is at two kinds of human cell lines' cell surface expression.Anti-Human CD3 scFv (G19-4) obtains expressing by the reverse transcription virus gene transduction at the cell surface of Reh and T51 clone.Can detect CH2 and CH3 structural domain among latter's detectable gene product IgG with corresponding fluorescence Anti-Human IgG in the G19-4 of cell surface expression scFv gene product.In each drawing, the reaction that takes place with wild-type cell dots, and represents with solid line with the reaction that transfectional cell takes place.

Figure 11. with cell surface expression anti--human T lymphocyte's of human cell line's co-cultivation of CD3 scFv amplification.This figure has shown and has cultivated the back inductive T of institute cell proliferation at the Reh of the anti-CD3 scFv of surface expression or T51 cell (but not wild-type Reh or T51 cell).In cultured cells, added in last eight hours a culture cycle that continues three days ³The H-thymus pyrimidine can be measured amplification.

Figure 12. immobilized human peripheral blood mononuclear cell cracking is from two cells the anti-CD3 scFv of cell surface expression human cell line.This figure has shown that the immobilized peripheral blood lymphocytes can kill Reh and the T51 cell at tumor cell surface expression Anti-Human CD3 scFv fast, but can not kill the Reh or the T51 cell of wild-type. ⁵¹The clone of Cr-mark and peripheral blood lymphocytes according to shown in ratio hatch 8 hours (in triplicate), measure d/d then ⁵¹Cr.The formula of available classics calculates the per-cent (the experiment burst size deducts spontaneous burst size, deducts spontaneous burst size divided by maximum burst size again) of specific killing.

Figure 13. when the ratio of K1735M2/500A2 cell and wild-type K-1735 cell is 1: 10, the wild-type K-1735 cell that can suppress to mix with it at the K1735-500A2 cell of cell surface expression Anti-Human CD3 scFv forms tumour, and this has confirmed " bystander effect ".This figure has shown to be had immunocompetently in body (C3H) mouse body, and the outgrowth of the mixture of K1735M2/500A2 cell and wild-type K-1735 cell (ratio is 1: 10) is suppressed.Independent immune wild-type K1735 cell, or wild-type K-1735 cell mixed with 10: 1 ratio mutually with the K1735M2/500A2 cell of transfection make the cell of untransfected transfected.With 2 * 10 ⁶Individual wild-type K1735 cell and 2 * 10 ⁶Individual K1735M2/500A2 cell mixes mutually, more mixed cell is used for subcutaneous immune C3H mouse.Every 5 days the growth of tumor situation is detected.

Figure 14. take from the splenocyte of the mouse of K1735-500A2 cellular immunization with can amplification after wild-type K1735 cell through irradiation combine, but when combining, then can not with the Ag104 cell.

Figure 15. be implanted into from the intravital K1735-1D8 cell of body mouse and can be ostracised, this is because CD4 ⁺Due to the common repulsion mechanism that produces of cell and natural killer cell.This figure has shown that the K1735-1D8 cell that is implanted into C3H mouse is by CD4 ⁺The mechanism of action that cell and natural killer cell produced repels.A) contain the virus vector structure of anti--mouse 4-1BB hybridoma 1D8 scFvDNA; B) use and phycoerythrin link coupled F (ab ') ²The anti-people's of goat immunoglobulin (Ig) can detect the expression of 1D8 scFv, this immunoglobulin (Ig) can be identified in the immunoglobulin (Ig) afterbody that express (shadow region) on the K1735-1D8 cell, but can not discern the immunoglobulin (Ig) (solid line) on the wild-type K1735 cell; C) K1735-1D8 (zero) cell and wild-type K1735 cell (■) cell are in the intravital growth kinetics of natural mouse; D) the intravital K1735-1D8 cell of mouse is by CD4 ⁺(■), CD8 ⁺(), CD4 ⁺And CD8 ⁺(zero) or the growth kinetics that exhausts of natural killer cell (△), in this experiment, give control group (●) injected in mice be mouse IgG behind the purifying.

Figure 16. use the K1735-1D8 cell, and need not carry out immunity, can resist the growth of transplanted wild-type K1735 cell, believe that this is because the memory and the specific mechanism of immunity cause through the wild-type K1735 of radiation exposure cell.A) by carrying out the method for subcutaneous transplantation with K1735-1D8 (zero) or through the wild-type K1735 of radiation exposure cell (), or with phosphate buffered saline buffer (PBS) (■) every ten days to subcutaneous immune twice of mouse (10/group).Immunity was given these injected in mice wild-types K1735 cell after ten days the last time, and carried out the immunity second time (▲) with wild-type cell once more for after two months the mouse with anti-K1735-1D8 immunity; B) with the Ag104 cell with 3 * 10 ⁵The concentration an of/mouse is transplanted to the mouse that has anti-K1735-1D8 immunity (■), and injects wild-type K1735 cell twice, to control group mice injection phosphate buffered saline buffer (PBS) (zero); C) to after having had the injected in mice wild-type K1735 cell of anti-K1735-1D8 immunity, its skin of back can decolour.

Figure 17. the K1735-1D8 cell can be carried out subcutaneous transplantation as vaccine, the wild-type K1735 tumour that growth forms in subcutaneous or lung is treated.This figure has shown with K1735-1D8 and as immunogenic the wild-type K1735 tumour that has formed has been treated.A) use hypodermic injection, reach 30 square millimeters mouse for wild-type K1735 tumour area, in the first six day of transplanting, the side is inoculated K1735-1D8 (zero) or is carried out immunity () through the wild-type K1735 of radiation exposure cell in tumour; Subcutaneous injection phosphate buffered saline buffer (PBS) mouse (■) is regarded as control group mice.Also to do identical treatment at given (↓) time point; B) give untreated mouse transplant wild-type K1735 cell 20 days after (left figure), or when accepting the K1735-1D8 immunity the 8th day the time first after accepting wild-type K1735 cell (middle figure), (right figure) will carry out immunohistochemical analysis to the tumour of collecting when injecting the 1D8 monoclonal antibody first the 8th day the time after accepting wild-type K1735 cell.The CD4 in the same tumor nodule has been represented in upper and lower two zones of every photo respectively ⁺And CD8 ⁺Cell; C) accept the tumour lung metastasis of intravenous injection wild-type K1735 cell mouse.Last figure has shown the lungs of control group mice, and figure below has shown transplants the lungs that the K1735-1D8 cell carries out mice immunized repeatedly.

Figure 18. the splenocyte of the K1735-1D8 immune mouse of hanging oneself can increase when combining with wild-type K1735 cell, but the Ag104 cell different with antigenicity in conjunction with the time then can not increase.This figure has shown the amplification through the K1735-1D8 immune mouse spleen cell.A) with taking from twice, and through the wild-type K1735 of radiation exposure (" K1735 ") or Ag104 (" Ag104 ") co-cultivation three days respectively with K1735-1D8 or through the splenocyte of the wild-type K1735 of radiation exposure cellular immunization mouse; The splenocyte that to take from natural mouse in contrast.Amplification to the tritiated splenocyte is measured; B) to using the CD4 of CFDA SE mark ⁺And CD8 ⁺Splenocyte carry out the flow cytometry analysis.

Figure 19. take from the splenocyte of K1735-1D8 immune mouse and can secrete interferon-, and have the activity of cytotoxic lymphocyte.This figure has shown the secretion and the cellular cytoxicity activity of interferon-.A) with behind K1735-1D8 cell twice immune natural mouse the 10th day, directly the ELISPOT that the splenocyte of taking from natural mouse is carried out interferon-analyzes.The splenocyte that last immunity is collected in the time of back 10 days joins in the interferon-ELISPOT culture dish, hatches 24 hours; To compare from the splenocyte of natural mouse with from the detected result that carries wild-type K1735 mice with tumor splenocyte; B) during 30 days carry out immunity with wild-type K1735 cell after, the situation of will be in experiment secreting interferon-at external (■) that is upset and unprovoked () splenocyte be summarized in the table 1.Following every group (three use handle with quadrat method) average spot number (from top to bottom) of mouse all shows: before handling 1 day with the K1735-1D8 cell, or after handling 4 or 8 days with wild-type cell; Before handling 1 day with the monoclonal antibody of 1D8, or after handling 4 or 8 days with wild-type cell.Below two row provided the ELISPOT data of the control group splenocyte of taking from natural mouse; C) take from the splenocyte of K1735-1D8 cellular immunization mouse with through the wild-type K1735 of radiation exposure cell co-cultivation 5 days, and detect cracked wild-type K1735 cell (■), Ag104 () and YAC-1 (zero) cell 4 hours ⁵¹The Cr release test, d) to do an experiment similar to Fig. 5 c, with except the splenocyte of having hatched jointly with anti-asialo GM1 antibody and rabbit complement, like this can with remove natural killer cell through the wild-type K1735 of radiation exposure cell co-cultivation with before detecting.Their the lysis activity that can resist wild-type K1735 cell (■) is suppressed by the monoclonal antibody (▲) of the anti-major histocompatibility antigen of I class.YAC-1 (zero) cell do not have cleaved in, can detect the lower dissolved cell activity of anti-Ag104 ().

Figure 20. at the K1735-1D8 cell of cell surface expression the anti-4-1 bb scFv, can be the mixture formation tumour that inhibition in 1: 10 o'clock is mixed with wild-type K1735 cell in the ratio of 1D8 and wild-type cell, this be proved to be a kind of " bystander effect ".This figure has shown to have immunocompetently in body (C3H) mouse body, and the growth of K1735-1D8 cell and wild-type K1735 cell mixture (ratio is 1: 10) is suppressed.Carry out immunity separately with wild-type K1735 cell, or with wild-type K1735 cell and the ratio of using the K1735-1D8 cells transfected with untransfected tumour cell and transfection tumor cell, promptly 10: 1 mixed immunity.With 2 * 10 ⁶Individual wild-type K1735 cell and 2 * 10 ⁵Individual K1735-1D8 cell mixes mutually, is used for subcutaneous immune C3H mouse.Every 5 days growth of tumor is monitored.

The anti-people 4-1BB of Figure 21 .5B9 scFv sequence.This figure has shown Nucleotide and the aminoacid sequence of the cell surface expression 5B9Ig of prediction.

Figure 22. table 1.

Figure 23. table 2.

Figure 24. table 3.

Figure 25. table 4.

Detailed Description Of The Invention

Unless stated otherwise, the present invention may adopt molecular biology (to comprise the restructuring skill Art), microbiology, cell biology, biochemistry, nucleic acid chemistry and immunologic multiple Technology, the technology under these all are included in the technical field. Various useful technology are at literary composition Explanation is all arranged in offering, for example, molecular cloning: laboratory manual, second edition (Sambrook et Al., 1989) and molecular cloning: laboratory manual, the third edition (Sambrook and Russel, 2001), (common and respectively reference conduct " Sambrook " at this); Oligonucleotides synthesizes (M.J. Gait, ed., 1984); Animal cell culture (R.I.Freshney, ed., 1987); Real Test immunology handbook (D.M.Weir and C.C.Blackwell, eds.); Mammal is thin Born of the same parents' gene transfer vector (J.M.Miller ﹠ M.P.Calos, eds., 1987); Molecular biosciences Learn up-to-date operation manual (F.M.Ausubel et al., eds., 1987, comprise whole year calendar year 2001 Supplementary issue); PCR: polymerase chain reaction (Mullis et al., eds., 1994); Immunology Up-to-date operation manual (J.E.Coligan et al., eds., 1991); Immunoassays handbook (D. Wild, ed., Stockton Press NY, 1994); Biological combination technology (Greg T. Hermanson, ed..Academic Press, 1996); The immunoassay method (R.Masseyeff, W.H.Albert, and N.A.Staines, eds., Weinheim:VCH Verlags gesellschaft MbH, 1993); Harlow and Lane (1988) antibody, laboratory manual, cold spring port Publishing house, New York, and Harlow and Lane (1999) uses antibody: laboratory manual, Cold spring port, New York publishing house, (at this common and respectively the list of references author be Harlow with Lane), Beaucage et al.eds, the up-to-date operation manual of nucleic acid chemistry, John Wiley ﹠ Sons, Inc., New York, 2000); And Agrawal, ed., oligonucleotides and class thereof Like thing synthetic operation handbook and characteristic Humana Press Inc., N.J., 1993).

Controlling of the disease that the present invention relates to benefit from antitumor or anticancer, not normal and illness Treat, prevent and/or alleviate, also relate to wherein used preparation.

The invention provides, for example, for generation of several different methods and the combination of antineoplastic immune Thing.

On the one hand, this invention provides a kind of new method, can be at external acquisition tumor response T lymphocyte group, this cell mass by with immobilization CD3 and independent CD28 or with CD28 adds PBMC and the tumour cell PBMC that the CD40 common combination stimulates common cultivation, The latter comprises patient with advanced cancer (overall immunosupress) from the cancer patient. The opposing party Face, for example, this invention provides composition, and it comprises and is coded in the anti-of cell surface expression The gene of CD3 scFv or anti-4-1BB scFv or other polynucleotides. On the other hand, this sends out The bright transfectional cell that to express these genes and be used for inducing antineoplastic immune that provides.

Stimulate and activating T cell with anti-CD3 and the CD28 antibody of immobilization on magnetic bead, all Can cause the growth of polyclone T cell and the generation of cytokine profiles (Levine et al., J Immunol, 159,5921-30,1997; Garlie et al., J Immunother, 22,336-45,1999). And breed through anti-CD3 and the post-stimulatory antigen of CD28 antibody from polyclone The generation of specificity T cell and/or the conversion of propagation are not formerly described.

As described here, for example,, especially in initial culture, add autologous tumor cell and can finish this conversion by at culture.The T cytoclasis that within 48-72 hour, is activated of test tumor cells showed, and the monocyte in the culture is in the same period reached maturity and is CD83 ⁺Dendritic cell.This is considered to it and is exposed to result in the lymphokine, and this class factor comprises interferon-and the tumor necrosis factor-alpha by activated T emiocytosis.Dendritic cell is engulfed the tumour cell of being eliminated, and the submission tumour antigen is given the activated T cell, constantly propagation and the growth of promotion tumour-specific T cell.

On the other hand, the invention provides gene or other polynucleotide of the anti-CD3 scFv of coding, this antibody fragment may be specific to CD3.This gene or polynucleotide can be transfected, are used for expressing such as the surface of people or mouse tumor cell system.In both cases, this cells transfected can activated T lymphocytes, makes its propagation, produces lymphocyte factor and killing tumor cell.

The in vivo test carried out in addition shows, can be had immunocompetent mouse at the mouse tumor cell of the anti-mouse CD3 of cell surface expression and repel, and induce the systemic immunity power of appearance to same tumour wild-type cell.Therefore, although can the inducing T cell activation when the tumor cell surface expression by the scFv of anti-mouse or anti-people CD3 genes encoding, the present invention confirms when the said gene product is used in vivo as a kind of tumor vaccine, and when tumour cell occurring in the body, this vaccine can change to antigen specific immune in the interior immunity of inductor.

On the other hand, gene that makes up among the present invention or other the polynucleotide for example anti-4-1BB scFv that can encode has specificity to 4-1BB, for example can be to the tumour cell of people and mouse with their transfections, and at these cell surface expressions.This transfected tumour cell can activate from the T lymphocyte of kind separately.Mouse tumor cell with anti-mouse 4-1BB scFv gene transfection can be had immunocompetent mouse repulsion, and this gene can be used as a kind of vaccine-induced tumour-specific immunity, repels the wild-type cell from identical tumour.This immune response has demonstrated memory and antigen-specific, and the tumour cell (K1735 melanoma) subcutaneous or that lung shifts that is grown in that is studied is had result of treatment.Because the I class major histocompatibility antigen molecular level that K1735 expresses is very low, and shortage II class major histocompatibility antigen molecule, and most human tumor cell's situation is also very similar, has low-down immunogenicity, so these results have important biological significance.

We have produced the gene of the coding scFv molecule that CD3 and 4-1BB with mouse and people can react.Each gene includes the tenuigenin tail of membrane spaning domain and people CD80.In addition, each gene all encode hinge area and human IgG1's CH2 and CH3 structural domain are between scFv binding site and membrane spaning domain.The cell of these genes and these gene transfections all is useful for the treatment tumour.

For example, in following experiment, pointing out, be coded in the anti-CD3 of cell surface expression or the cDNA of anti-4-1BB scFv molecule and can be used as a kind of DNA plasmid transduction, perhaps can in such as virus or bacteria carrier, transduce to tumour patient.External, be coded in the anti-CD3 of cell surface expression or the cDNA of anti-4-1BB scFv molecule and can be imported into tumour cell, the tumour cell after the transfection can be used for the treatment of.Can use from tumour cell body or allochthonous.The combination of the scFv gene coding molecule of cell surface expression comes from the combination of its encoding gene, and the expressed scFv molecule and the receptors bind of T cell surface provide activation or costimulatory signal.Also imagined another among the present invention and conducted the method that the polyclone activation signals is given the T cell in vivo, comprised to patient infusion comprising by the antibody of the surface receptor of T cell expressing or the slowly-releasing polymer of part.

The present invention also provides a kind of new method, at external generation/amplification tumor-selective T lymphocyte, the signal of these cells in the autologous tumor cell and CD3 and costimulatory molecules mediation occurs by activation, and the being subjected to property that is used for for example continuing is transferred to tumour patient.The present invention help can submission tumour cell released antigen in external generation dendritic cell, enlarging the tumor response T cell mass that has existed, and help producing previous unrecognized at antigenic immune response.At the T of external generation cell, more insensitive to the restraining effect of TGF-β as described in invention, and long life cycle is arranged.

The present invention has also described two kinds of novel human tumor vaccines that produce by the scFv gene of transfection encoding antibody derived molecules, they can discern CD3 or 4-1BB, lower and only express I class major histocompatibility antigen molecule on a small quantity and when not expressing the murine melanoma of II class major histocompatibility antigen molecule in their opposing immunogenicities of check, show that these vaccines can be induced to produce the tumor destruction immune response.This method of describing among the present invention can be used for the transfection human tumor cell, expresses anti-people CD3 or anti-people 4-1BB scFv and is used for immunity based on cell.In addition, it can more easily be used to make up the tumor vaccine based on gene, wherein the tumour epi-position of genes encoding can be with the anti-CD3 scFv of genes encoding or/and anti-4-1BBscFv combine.The gene that raises the lymphocyte factor of anti tumor immune response by scFv and/or coding in conjunction with that can discern other or different immunologic stimulant acceptors can make range of application of the present invention enlarge.

The present invention is further discussed with reference to specific experiment, and the mode of embodiment may be only adopted in these experiments, limits the invention never in any form.

Embodiment 1

The lymphocytic activation of tumor response from the CD3 of patient with advanced cancer mediation

Peripheral blood lymphocytes (PBMC) or tumor infiltrating lymphocyte (TIL), in coupling under the situation about existing with the magnetic bead of CD28 monoclonal antibody or CD28 and CD40 monoclonal antibody bonded CD3 monoclonal antibody, with the autologous tumor cell co-cultivation.The certain situation that takes place in cultivating is characterized, comprise that the tumour cell of cultivation is destroyed, the generation of lymphopoiesis and lymphocyte factor, CD83 ⁺The lymphocytic activation of the generation of antigen presenting cell and tumor response cytotoxic/amplification, and lymphocyte descends to the inhibiting susceptibility of TGF-β.

Take from patient's material.Tumour is taken from IV phase cancer patients's operation or pernicious exudate (most is ascites).Tumour and peripheral blood sample all obtain under informed consent.Most studies adopts 8 patients, wherein 5 (1OV, 3OV, 8OV, 44OV 48OV) has ovarian cancer, and 2 (1C 22C) suffers from colorectal carcinoma, and one (1HN) suffers from tumor of head and neck.The cell from ovarian cancer cell line 4007 has also been used in experiment.

The preparation of tumour and blood sample.Noumenal tumour is suspended from the substratum, removes the liquid portion in the exudate, then cell is overhang.(PharmaciaBiotech, Upsala Sweden) remove red corpuscle, utilize Percoll gradient (Sigma, St Louis, MO) separating tumor cell from tumor infiltrating lymphocyte with Ficoll-Hypaque.The lymphocyte sample can directly use or be stored in the liquid nitrogen in order to use in the future.The operation steps of use standard is transplanted to tumor sample external and is set up clone.Peripheral blood lymphocytes comprises the T lymphocyte, and monocyte and B cell carry out purifying with Ficoll-Hypaque to them.In a lot of initial experiments, used CD8 ⁺T lymphocyte (purity＞90%), these cells are that (Miltenyi Biotech company, Auburn CA) screens from tumor infiltrating lymphocyte application VarioMac magnetic bead.

The preparation of the magnetic bead of bind lymphocytes, antibody coupling and the culture of tumour cell.In initial experiment, 5 lymphocytes have been added in each tumour cell, mixture is being equipped with RPMI substratum and 10% foetal calf serum (Atlanta Biological then, Norcross, GA) 12-well culture plate (the Coming Inc. of Costar (3513), Coming, NY) in, in 37 ℃ environment, hatch.Subsequently, use a kind of disclosed technology (Levine, Bernstein et al., J Immunol, 159,5921-30,1997; Gariie, LeFever et al., J Immunother, 22,336-45,1999), with peripheral blood lymphocytes or tumor infiltrating lymphocyte and or not be coupled at magnetic bead (Dynal Inc., Lake Success, NY) the autologous tumor cell co-cultivation on, coupling has CD3 on the magnetic bead, CD28, and/or the monoclonal antibody of CD40; The magnetic bead that will not have a coupling monoclonal antibody (or coupling has incoherent monoclonal antibody) in contrast.The link coupled monoclonal antibody is respectively 64.1 (Martin et al., JImmunol, 136,3282-7,1986) (Martin, Ledbetter et al., J Immunol, 136,3282-7,1986), 9.3 (Martin, Ledbetter et al., J Immimol, 136,3282-7,1986) and G28-5 (Ledbetter et al., J Immunol, 138,788-94,1987), they stimulate lymphocyte (anti-CD3) in polyclone ground respectively, stimulate (anti-CD28), perhaps active antigen presenting cell (anti-CD40) altogether.When using autologous tumor cell, cell (40,000-75,000/ hole) at first needs to be adsorbed onto on Costar 24 well culture plates by night incubation, is added with the 2ml IMDM substratum that contains 10% foetal calf serum in the culture plate.Add monoclonal antibody coupling magnetic bead (3 * 10 then ⁶/ ml), adding is suspended in the lymphocyte (10 that contains among the 10% foetal calf serum RPMI again ⁶/ ml).Culture plate contains 6%CO at 37 ℃ ₂Air in hatched 4-5 days.Use magnet to remove magnetic bead, lymphocyte is placed a new interleukin-2 that contains 10U/ml, and (Roche Molecular Biochemicals, Indianapolis in culture plate IN), work as concentration then and reach 2 * 10 ⁶Behind individual cell/ml substratum is moved in the narrow-necked bottle.Observe substratum and seek the ruined evidence of tumour cell.Determine lymphocytic amplification situation by cell counting.From substratum, take a sample, use WEHI cell (Espevik and Nissen-Meyer respectively, J Immunol Methods, 95,99-105,1986) use a kind of bioanalysis and measure the tumour necrosis factor that produces, with ELISA (EH-EFNG, Endogen, Wobum, MA) method is measured interferon-.TGF-β 1 available from Sigma company (St Louis, MO).All can use TGF-β 1 in all tests, even if after having removed monoclonal antibody link coupled magnetic bead, these molecules are still stayed in the substratum.

The detection of cytotoxic lymphocyte.Used classical 4-hour ⁵¹The Cr method for releasing detects.For the on-effect cell qualitative, when experiment, in cell, add framework determinant (the Research Diagnostics Inc. that can discern I class major histocompatibility antigen molecule, Flanders, monoclonal antibody w6/32 NJ) (10ug/ml) suppresses cytotoxicity.Also can use the monoclonal antibody (BeckmanCoulter of natural killer cell sign antigens c D16 and CD56, Brea, CA), anti-CD8 monoclonal antibody HIT8a (BD Pharmingen, Lexington, KY), with anti-monoclonal antibody 60.3 (Beattyet al., J Immunol, 131 of integrating element-β 2 (CD18), 2913-8,1983).

Lymphocytic facs analysis.By FACS (Epics XL, Coulter, Miami, FL) the expression density of method assessment CD is used the monoclonal antibody of PE mark, and when cell reaches default minimum brightness they is counted the positive.In order to study whether lymphocyte CD3 expression density increase after external activation is causing owing to initial CD3 high expressing cell selective proliferative, with peripheral blood lymphocyte (the den Haan et al. of dyestuff CFDA to collecting from tumour patient, J.Exp.Med., 192,1685-1695,2000) (MolecularProbes, Eugene OR) carry out mark.Subsequently, in the nutrient solution that is attached with anti-CD3/CD28/CD40 magnetic bead, cultivated 5 days, remove magnetic bead then, the lymphocyte after the amplification is moved in the substratum that contains 10U interleukin-2/ml.Carry out facs analysis during two time points (4 hours and 3 days) after removing magnetic bead, analyze the CFDA brightness in each hole and the expression of CD3.The lymphocyte that is labeled after cultivating is used for comparative studies with contrast magnetic bead co-cultivation.

The antibody coupling magnetic bead stimulates T cell low reaction level.Begin to have carried out 6 experiments, the CD8 of purifying from tumor infiltrating lymphocyte ⁺T lymphocyte and tumour cell co-cultivation, suspension is carried out the analysis of tumour necrosis factor or interferon-.In a representational experiment, will take from a colorectal carcinoma patient, 1C, CD8 ⁺Tumor infiltrating lymphocyte with 1C tumour cell co-cultivation 15 days, takes out lymphocyte then, and adds in one group of fresh 1C cell or add the tumour cell of taking from a patients with lung cancer--among the 3L.When the 1C lymphocyte combines with 1C and when not combining with 3L, only record a spot of tumour necrosis factor (1.2pg/ml), but when combining with the 3L tumour cell, but when not having single culture, tumour necrosis factor and interferon-(1.5pg/ml) have been produced from the tumor infiltrating lymphocyte of 3L.There is not lymphopoietic evidence.In experiment subsequently, will be except that CD8 ⁺Outside the lymphocyte, also include monocyte, CD4 ⁺The tumor infiltrating lymphocyte cell mass of T cell and B cell was with autologous tumor cell co-cultivation 10-15 days.In 13 patients, have in 8 patients' the cultivation suspension to have detected about 10 times of high-caliber tumour necrosis factors (4.5-48pg/ml) and interferon-(up to 150pg/ml).Still there is not lymphopoiesis.

When autologous tumor cell and anti-CD3 coupling magnetic bead co-cultivation, the demonstration of T cell proliferation and tumor cell destruction.Carry out system's experiment after the initial experiment, promptly use monoclonal antibody link coupled magnetic beads by various lymphocyte receptors (Levine, Bernstein etal., J Immunol, 159,5921-30,1997; Garlie, LeFever et al., JImmunother, 22,336-45,1999) induce the generation signal.Have in the substratum of CD3 monoclonal antibody and CD28 and/or the existence of CD40 monoclonal antibody magnetic bead, in coupling peripheral blood lymphocytes or tumor infiltrating lymphocyte and autologous tumor cell co-cultivation.Also comprise the same packets that contains lymphocyte but do not contain tumour cell.In contrast, the lymphocyte that contains or do not contain tumour cell with without link coupled contrast magnetic bead co-cultivation.After 3-5 days, remove magnetic bead, lymphocyte and tumour cell were hatched respectively in the interleukin-2 of 10U/ml 2-21 days.

Fig. 1 shows the experimental result of vigorous propagation when OV44 patient tumors lymphocyte infiltration is cultivated 4 days in anti-CD3/CD28/CD40 coupling magnetic bead.The lymphocyte that lacks the CD3 token stimulus can not bred, and the lymphocyte that uses anti-CD28 and/or CD40 magnetic bead to cultivate also can not be bred (not display data).Propagation more (drawing B) during the initial magnetic bead co-cultivation with by the CD3 inducement signal of autologous tumor cell.Anti-CD3/CD28 coupling magnetic bead inductive propagation and anti-CD3/CD28/CD40 coupling magnetic bead identical (data not shown).

Fig. 2 shows peripheral blood lymphocyte and various monoclonal antibody coupling magnetic bead and autologous tumor cell or 5 days the experimental result of allogeneic (4007) cell cultures from patient 1C.When CD3/CD28 (drawing C) or anti-CD3/CD28/CD40 (Fig. 2 D) activated lymphocytes and 1C tumour cell co-cultivation, the quantity of each culture hole medium size lymphocyte height during than itself and 4007 cell co-cultivation, the result is identical to those shown in Fig. 1.Facs analysis shows that＞90% activated lymphocytes is expressed CD3, and about 70% is CD8 ⁺, be less than 5% and express CD16 or CD56.On the other hand, do not provide at magnetic bead under the situation of signal of any CD3 mediation (Fig. 2 A and B), add the allos tumour cell after the propagation level higher, may be because the isoantigen of expressing on 4007 cells is produced immune response.

Most of tumour cells with the situation that has anti-CD3/CD28 or anti-CD3/CD28/CD40 coupling magnetic bead under cultivate contact within 24-48 hour destroyedly from the body lymphocyte, usually can stay and contain fully by the substratum that the cellularity of lymphocyte form is arranged.Whether have immunologic opsonin in order to study this tumor destruction, carried out four experiments, at first to peripheral blood lymphocyte (10 from tumour patient ⁶-10 ⁵/ increment this) carry out gradient dilution, with them and autologous tumor cell or from the tumour cell or the fibroblast co-cultivation of allogeneic donor.In two experiments, with have about tumour necrosis factor more than 10 times in the cell suspension of autologous tumor co-cultivation, but from not having difference on the data in killer cell between body or allogeneic tumour cell or the allogeneic fibroblast co-cultivation base.Inferring that thus the destruction of the tumour cell that occurred after lymphocyte activation 24-72 hour does not have antigen-specific, perhaps is because a large amount of activated T lymphocyte and lymphocyte factor can not be distinguished specific component.

The generation of tumor-selective cytotoxic lymphocyte.The restrictive cytotoxic lymphocyte of I class major histocompatibility antigen adds that by tumour cell anti-CD3/CD28 or anti-CD3/CD28/CD40 magnetic bead activated lymphocytes produce.Fig. 3 shows an experiment of being made of the peripheral blood lymphocyte of taking from patient 1C (being activated) in the experiment that Fig. 2 shows, after tumour cell and the activation of monoclonal antibody coupling magnetic bead, magnetic bead is removed, and lymphocyte amplification cultivation in the 10U interleukin-2/ml substratum that lacks tumour cell and magnetic bead was surpassed for 3 weeks.By 1C and anti-CD3/CD28 magnetic bead activatory peripheral blood lymphocyte the 1C cell is had very strong cytolysis, CD8 monoclonal antibody and I class major histocompatibility antigen monoclonal antibody w6/32 can suppress this solvency action (Fig. 3 A).50: 1 E/T only can kill 20% allogeneic 4007 cells, and 98% 1C cell can be by 50: 1 E/T dissolving (Fig. 3 A) by contrast.Fig. 3 B provides the class likelihood data of the peripheral blood lymphocyte that is stimulated by anti-CD3/CD28/CD40 magnetic bead.The 1C that the dissolving of 4007 cells is cultivated when having monoclonal antibody w6/32 is in same low level.By contrast, anti-CD3/CD28/CD40 magnetic bead activatory peripheral blood lymphocyte can kill and wound 1C and 4,007 two kinds of cells, and this situation also appears under the situation that CD8 monoclonal antibody or monoclonal antibody w6/32 exist (data not shown).Be presented at the concentrated CD8 that obtains from cell mass that uses in the experiment among Fig. 3 B ⁺Cell with 20/1 E/T ratio solvent 25% 1C cell, compare and dissolved 0% cell from 4007 clones, also dissolved 0% cell from allogeneic B clone.In this experiment, the dissolution rate that uses monoclonal antibody w6/32 to cultivate the 1C cell is 5%, dissolution rate when using anti-CD18 monoclonal antibody 60.3 to cultivate is 5%, uses CD16 and CD56 monoclonal antibody to unite when cultivating, and dissolution rate only reduces to 18% from 25%.By can't optionally dissolving 1C or 4007 cells with 4007 cells and any magnetic bead combined cultivation activated lymphocytes.Repetitive cell toxicity lymphocyte analytical method twice all obtains same result.

The generation of Th1 type lymphokine.Visible detection is to a large amount of interferon-s in the CD3 mediation activated lymphocytes culture supernatant.Among Fig. 4 this is illustrated, this illustrates that also the interferon-that produces when also having autologous tumor cell during 4-5 days of cultivation beginning is higher.

Table 1 shown six extra, representational experiment shows being created in the cell of taking from the patient or all can being detected of the propagation of peripheral blood lymphocyte or tumor infiltrating lymphocyte and lymphocyte factor after the external magnetic bead activation that takes turns.Anti-CD3, anti-CD3/CD28, anti-CD3/CD40 and anti-CD3/CD28/CD40 magnetic bead all can promote lymphocytic propagation consumingly, the effect between them does not have difference.Comparatively speaking, use anti-CD28 separately, anti-CD40 and anti-CD28/CD40 magnetic bead more can increase the generation of lymphocytic propagation and lymphokine unlike contrasting magnetic bead, and this signal that shows the CD3 mediation is extremely important.The generation of tumour necrosis factor and interferon-has dependency.Growth is in the time of 3-5 days under the situation of tumour cell and magnetic bead when lymphocyte is not having, and their content is reduced to background level.As shown in figs. 1 and 4, induce effective lymphopoiesis and lymphokine to produce and need the CD3 signal.

Rise by CD3 on monoclonal antibody-coupling magnetic bead activated lymphocytes and other marks.Before tumour cell and anti-CD3/CD28/CD40 magnetic bead are cultivated 3 to 5 days and afterwards, measure the expression density of T cell differentiation antigen on lymphocyte populations with FACS, then 3 to 7 days then for not adding the amplification phase that magnetic bead is cultivated.In order to react the variation of CD expression of receptor density, report the per-cent of each group cell, the set(ting)value brightness that their density equals to select perhaps is higher than set(ting)value (table 2); The peripheral blood lymphocyte that analysis is not stimulated from 6 healthy donors (age was at 30 to 65 years old) as a comparison.From the CD3 of the peripheral blood lymphocyte that is not stimulated of tumour patient, the level of CD4 and CD28 is lower.4 CD3 density among 5 patients are also lower, and the density of CD86 is higher than the peripheral blood lymphocyte level that healthy donor is not stimulated.Express the part increase with the peripheral blood lymphocyte CD3 of contrast magnetic bead co-cultivation, but the expression of CD28 increase is not obvious.Under comparing, use the expression amount of anti-CD3/CD28/CD40 magnetic bead cultured cells CD3 and CD28 can both return to normal level, and the cell quantity that makes high CD8 express density double.5 patients' TIL is studied the expression density of CD3.Be respectively 2.9%, 40.2%, 96%, 42.8% and 40.1%, show also more changeable, and usually above peripheral blood lymphocytes.The CD3 of tumor infiltrating lymphocyte expresses and is higher than peripheral blood lymphocytes, and is increased to 87.3% from 61.4%.The analog value that CD28 expresses in the tumor infiltrating lymphocyte is 39.3% and 52.8%.

In order to study whether the increase that lymphocyte CD3 after external activation expresses density is because the value-added result of cell selective that initial high CD3 expresses, can use dyestuff CFDA (Molecular Probes, Eugene OR) carries out mark to concentrating the peripheral blood lymphocyte that obtains from tumour patient.The use tumor infiltrating lymphocyte experimentizes, and the 40.2% initial CD3 that expresses uses dyestuff CFDA (den Haan, Lehar et al, J.Exp.Med., 192,1685-1695,2000) and carries out mark.After anti-CD3/CD28/CD40 magnetic bead activation, CD3 expresses and increases to 95%.Use the anti-CD3 of CFDA and PE-mark to carry out facs analysis as probe, the result shows the selective proliferative that does not have initial CD3 high expression level lymphocyte subpopulation.

The expression of the CD83 of culturing cell after using anti-CD3/CD28 magnetic bead activation.Use anti-CD3/CD28/CD40 magnetic bead to stimulate peripheral blood lymphocytes (finding that wherein 10-20% expresses monocyte mark CD14) whether can promote the maturation of dendritic cell in order to study, the different time points after stimulating is measured the expression of CD83.CD83 is expressed by the dendritic cell after the maturation, and immature dendritic cell and blood monocyte are not expressed.Fig. 5 has illustrated a typical experiment, shows 24 hours after using anti-CD3/CD28 magnetic bead stimulation PBMC, just can detect the expression of CD83 in 35% cell.Do not detect the expression of CD83 in the 0th day (before the stimulation) stimulating peripheral blood lymphocytes.

In order to determine peripheral blood lymphocytes stimulates what cell expressing CD83 afterwards, using after stimulating the 2nd day of anti-CD3/CD28 magnetic bead or anti-CD3/CD28/CD40 magnetic bead, use fluorescein-labeled anti-CD3 that two kinds of colors of anti-CD83 of PE-mark are dyeed.Fig. 6 is presented at and lacks the expression that the magnetic bead stimulated cells does not have CD83, and anti-CD3/CD28 coupling magnetic bead is induced the expression of CD3 negative cells group (13.9%) CD83, and suitable ratio expression CD83 is also arranged in the CD3 positive cell.By contrast, anti-CD3/CD28/CD40 coupling magnetic bead can activate the expression of inducing CD83, and is lower than inducing of anti-CD3/CD28 magnetic bead but institute's inductive CD3 negative cells and CD3 positive cell are expressed the level of CD83.These results show, the cell of expressing dendron shape cell sign thing CD83 after stimulating through anti-CD3 and anti-CD28 monoclonal antibody coupling magnetic bead from the peripheral blood lymphocytes rapid induction.Anti-CD3, the hormesis of anti-CD28 and anti-CD 40 monoclonal antibody coupling magnetic bead, not anti-CD3 and anti-CD28 monoclonal antibody coupling magnetic bead are used to stimulate the expression of CD83 effective like that separately.

The activation signals that produces by monoclonal antibody coupling magnetic bead increases TGF-β 1 inhibiting resistance.Table 3 has shown 5 representational experiments, and 1 pair of lymphokine of research TGF-β produces and whether lymphopoietic inhibition effect can be by being changed with the magnetic bead co-cultivation that can induce CD3 mediation signal.Use the contrast magnetic bead, tumour necrosis factor and interferon-level are very low, and their level is further suppressed by TGF-β 1.By contrast, when using anti-CD3/CD28/CD40 magnetic bead, their level has been increased to the level that occurs when not having TGF-β 1.Equally, when using anti-CD3/CD28/CD40 magnetic bead, TGF-β 1 is littler for lymphopoietic restraining effect, at patient 1HN even do not find restraining effect at all.When the dosage of TGF-β 1 is increased to 20ng/ml, and reduce to 10 when lymphocytic concentration ⁵During/sample (data not shown), also can see the propagation of T cell and the generation of lymphokine and relative repulsion occur.When magnetic bead passes through CD28, when CD40 stimulates separately or jointly, can't resist the effect (data not shown) of TGF-β 1.

Therefore, be accompanied by killing and wounding of tumour cell, caused antigenic release with the lymphocyte activation of tumour cell co-cultivation.Monocyte in the substratum is engulfed tumour antigen, is divided into CD83 male antigen presenting cell submission antigenic determinant, makes tumor response T cell selective propagation.The treatment vaccine can be based on same principle, and activation and propagation tumour are carried the downtrod lymphocyte in the individuality, also can promote the immunoreactive generation at the subdominance antigenic determinant.In general, the culture system of generation tumor response T cell will comprise four parts.They are:

1) derive from cancer patients's T cell,

2) from patient's antigen presenting cell,

3) with anti-CD3 and anti-CD28 antibody, or anti-CD3, anti-CD28 and anti-cd40 antibody link coupled magnetic bead and

4) from patient's tumour cell

These integral parts can have a variety of variations, this down variation can be predicted or estimate.Comprise the variation of any four kinds of integral part joining days, also have the difference in four kinds of integral part sources.For example, patient T cell can separate from peripheral blood or tumor infiltrating lymphocyte.Antigen presenting cell is present in an embodiment in the peripheral blood lymphocytes part, but other sources, for example marrow can be arranged.What use in substratum among the embodiment is autologous tumor cell, but also can add allogeneic tumour cell or tumour antigen or with its alternative autologous tumor cell, this is to come submission by the self antigen presenting cell because of tumour antigen.Anti-CD3 and anti-CD28 antibody coupling magnetic bead can be by the immobilised antibody surrogates of other approach, also can promote the activation of polyclone T cell and the propagation of tumor response T cell by forming at immobilization antibody or specific part that other cell surface receptors produced.

These working method also make the generation of CD3 positive lymphocyte become possibility, continue to surpass for 10 weeks in vitro culture, promptly can be used for adoptive immunotherapy.This may be possibility (Boise et al..Immunity, 3,87-98,1995 of having reduced Lymphocyte Apoptosis owing to by the combined stimulation of CD28; Daniel et al., J Immunol, 159,3808-15,1997), prolong (Levine, Bernstein et al., J Immunol, 159,5921-30,1997) in external life cycle.Prolong different with lymphocyte survival time under the situation about existing at the high dosage interleukin-22, the lymphocyte that stimulates also can prolong (Ranga et al., Proc.Natl.Acad.Sci., 95 at the patient's time to live after body of transduceing back altogether, 1201-1206,1998).It should be noted that, the hormesis of the T cell of CD3 mediation combines with the independent combination of CD28 or with CD24 and finishes, can prevent the restraining effect that lymphopoiesis and tumour necrosis factor and interferon-are produced of about 50% TGF-β 1 mediation, even when the concentration of TGF-β 1 in nutrient solution of use is in the saturated level of 20ng/ml, also be like this.

Embodiment 2

The structure that contains the carrier of anti-people of coding and anti-mouse CD3 scFv, transfection, but and confirm to stimulate at the polyclone of the anti-CD3 scFv of cell surface expression inducing T cell, make its propagation, and produce Th1 type lymphokine, have cytolytic and anti-tumor activity in the body

Above-described experiment shows that in the substratum that contains autologous tumor cell and peripheral blood lymphocytes or tumor infiltrating lymphocyte, the signal that is provided by CD 3-resisting monoclonal antibody coupling magnetic bead makes tumor response lymphocyte activation and amplification.Here the gene of mentioning is meant the gene that is used for oncotherapy of structure, can have active CD 3-resisting monoclonal antibody scFv (scFv) derivative at tumor cell surface expression.Made up with mouse CD3 with hybridoma 500A2 and to have had reactive anti-CD3 scFv, made up with people CD3 with hybridoma G19-4 and have reactive anti-CD3 scFv (Ledbetter et al., J.Immunol., 136,3945-3952,1986).

The structure of scFv.By cloning from hybridoma RNA (Hayden et al., TherImmunol, 1,3-15.1994; Gilliland et al., Tissue Antigens, 47,1-20,1996) antibody variable region light chain and the heavy chain cell surface configuration that makes up scFv (scFvs).Hybridoma is grown in the RPMI solution [10% foetal calf serum, 4mM glutaminate, 1mM Sodium.alpha.-ketopropionate and 50u/ml mycillin, (all available from Life Technologies, Gaithersburg MD)], keeps logarithmic growth mode a couple of days before harvested cell.By centrifugal suspending nutrient solution collecting cell, use Trazol or use the extractive method of QIAGEN RNA post (LifeTechnologies, Gaithersburg MD, and QIAGEN, Valencia, a kind of NP-40 dissolving technology (Gilliland of improvement is perhaps used in the CA) explanation that provides according to the manufacturer, Norris et aL, Tissue Antigens, 47,1-20,1996), from 2 * 10 ⁷Extract RNA in the individual cell.(Japan), and total RNA of 1 microgram carries out synthetic cDNA first chain of random priming for Takara Shuzo, Otsu Shiga to use Superscript II ThermoScript II (Life Technologies) and random hexamer.Behind reverse transcription, make the cDNA fragment have poly G tail with dGTP and terminal enzyme (DNA), this kind of enzyme can the catalysis triphosphate deoxy-nucleotide 3 of a DNA chain end '-the OH group adds a deoxynucleotide.CDNA has the grappling tail, can increase the mRNA clone's who at one end has unknown leading peptide efficient.As described, 5 ' end primer is the ANCTAIL primer of a modification, comprises a poly C tail, is used for polymerase chain amplified reaction (the Loh et aL of TXi Baoshouti nucleic acid chains sequence, Science, 243,217-20,1989), it also has SacI, and conveniently clone in XbaI and EcoRI site.This sequence is as follows:

5’-cgtcgatgagctctagaattcgcatgtgcaagtccgatgagtccccccccccccc-3’

3 ' the end primer of cDNA is from the constant region of heavy chain or light chain, and about 50 bases of J-C joining region are crossed in combination.Each 3 ' end primer includes HindIII, BamHI and SalI cloning site.Therefore, be used for the part of the restriction site of initial fragment subclone as these initial pcr amplification primers, digest amplification PCR fragment and subclone be to pUC19, pSL1180, or the TOPO carrier (Invitrogen, San Diego CA) are used for order-checking.Use pUC, T7, or Ml3 is general and reverse primer, and terminal cycle sequencing test kit (the PE Biosystems that ends of BigDye, Foster City, CA), (QIAGEN.Valencia CA) carries out dna sequencing to miniprep DNA with ABI Prism 310 (PE Biosystems) sequenator.

In case be separated to the variable region and obtained identical sequence at least three identical clones, just can use overlapping oligonucleotide to make up scFv by pcr amplification, cause the cDNA of coding light chain and variable region of heavy chain to merge.Add a kind of (gly4ser) ₃Connexon makes light chain connect together (Gilliland, Norris et al., Tissue Antigens, 47,1-20,1996) with variable region of heavy chain during this stitching polymerase chain reaction as the method for the part of overlapping oligonucleotide.The scFv molecular subcloning that assembling is finished is to merging human IgG1's hinge area that people CD80 strides film district and tenuigenin tail, the upstream of CH2 and CH3 structural domain (Winberg et al., Immunol Rev, 153,1996) according to reading frame.The natural guiding peptide chain in complete expression cassette coding light chain V zone, or come the secretion signal peptide of L6VK light chain of the SalI site fusion of self energy and scFv variable region.ScFv is by HindIII-BclI, or SalI-BclI box coding, and its first restriction site conforms to the opening frame of reading, and meanwhile, second restriction site is outside the encoder block relevant with fusion rotein.The human IgG1's wild-type Fc structural domain that then this box and coding region is positioned on the BstBI-ClaI section merges mutually.The method that afterbody in diaphragm area and the tenuigenin all can pass through polymerase chain reaction (PCR) of striding of CD80 is come from human tonsil RNA amplification, and its coding region is positioned on the BstBI-ClaI section, comprising a terminator codon that just is in upstream, ClaI site.Each subfragment is all used in the middle of the multi-link son/multiple clone site of method insertion artificial of subclone, these sites have been inserted in the middle of the modified pCDNA3 carrier in advance.In case the complete fusion rotein structure assembling of Codocyte surface scFv finishes, just whole expression cassette can be transferred to (Miller and Rosman in the middle of the reverse transcription expression vector pLNCX as a kind of HindIII-ClaI fragment, Biotechniques, 7,980-2,984-6,989-90,1989) (Fig. 9).

Transfection.Plasmid DNA is to be come by the reverse transcription preparing carriers of reorganization, and uses CaPO ₄The two preferendum packing cells of precipitation technology (Winberg, et aL, (1996) Immunol Rev.153:209-223.) transfection PT67 (Clontech, Palo Alto, CA).In brief, with cell with about 25% degrees of fusion, add and to contain the nonessential amino acid of DMEM of 10% foetal calf serum, 4mM glutaminate, 2 times of concentration and the DMEM substratum of penicillin-Streptomycin sulphate (this prescription is called as DMEM-C hereinafter, all reagent is all available from LifeTechnologies company) in, overnight incubation before transfection.Plasmid DNA is added the CaCl that 0.5ml concentration is 0.25M ₂In the solution, dropwise add the HEBS damping fluid (pH 7.1) of 2 times of concentration of 0.5ml again.Under 37 ℃ environment, can separate out precipitation after 5 minutes, then solution is dropwise added be grown in be added with DMEM-C substratum (8ml), diameter is in the cell in the culture dish of 100mm.To use phosphate buffered saline buffer (PBS) to clean twice then through the cells transfected overnight incubation, pour fresh substratum again into.From transfected cell, collect viral supernatant, and after 24 hours, use it for transduction.Perhaps, will be through transfection, adherent PT67 cell that can express cell surface scFv and cell co-cultivation in suspension of B clone.After going down to posterity several times,, carry out Tao Xuan with the B clone of the anti-human IgG1's pair cell of the goat of immobilization on culturing bottle surface expression scFv with the dilution of the packing cell in the substratum.The fixation degree of cell on culturing bottle of expressing high-level cell surface scFv is comparatively firm, and can not express cell this antibody or that the antibody expression level is lower can be by wash-out from culturing bottle.Be used in method that the culturing bottle surface scrapes cellular segregation, before being used for biological analysis, cultivate a few days again high level expression.

Mouse and tumor cell line.Bought the old female C3H/HeN mouse (Taconic, Germantown, New York) in 6 to 8 weeks.K1735 is the malignant melanoma that a kind of C3H/HeN takes place, and has selected a kind of transfer clone M2 (Fidler and Hart, CancerRes, 41,3266-3267,1981).Consistent with previous discovery, find its MHC I class expression very low (data not shown).Animal facility be through ALAC authentication and experimental technique approve through corresponding public animal association.

Antibody.R-phycoerythrin (PE)-coupling monoclonal antibody GK1.5 (anti-mouse CD4), AF3-12.1 (the anti-H-2K of 53-6.7 (anti-mouse CD8a) and purifying ^K) available from Pharmingen company (San Diego, California), R-PE link coupled goat F (ab ') ₂Anti-human IgG available from Biosource International company (Camarillo, California).Monoclonal antibody 169-4 (anti-CD8) from doctor R.Mittler (Emory University, Atlanta, Georgia).GK1.5 (anti-CD4) produces from ATCC hybrid cell knurl.

The transfection of carrier and K1735 cell.Described the variable region clone, scFv makes up and scFv expresses method (Gilliland, Norris et al., Tissue Antigens, 47,1-20,1996 that produce; Hayden et al., Tissue Antigens, 48,242-54,1996; Winberg, Grosmaire et al., Immunol Rev, 153,1996).The present invention adopt the gene from anti-CD3 hybridoma 500A2 or anti-4-1BB hybridoma 1D8 variable region make cell in conjunction with 500A2 scFv or 1D8 scFv at cell surface expression (Hayden, Grosmaire et al..Tissue Antigens, 48,242-54,1996; Winberg, Grosmaire et al., ImmunolRev, 153,1996).In order to express scFv, used membrane spaning domain and tenuigenin tail from CD80 because its mediated cytoskeleton adhere to cell and cells contacting the time crosslinked (Doty and dark, J Immunol, 157,3270-9,1996; Doty and Clark, JImmunol, 161,2700-7,1998).The scFv gene fusion in pLNCX, is passed through CaPO ₄Intermediate processing transfection RetroPack ^TMThe PT67 packing cell (ClontechLaboratories.Inc, Palo Alto, California).Use is from the substratum transfection K1735-WT cell of these cells.Use the goat anti-human igg of PE mark that the G418 resistance clone is carried out the expression that mark is used for the scFv cell surface.

Zooscopy.Mouse, 5 or 10/group, back one side subcutaneous transplantation 2 * 10 ⁶(12,000 rad) wild-type K1735 cell of individual wild-type K1735 cell or K1735-500A2 cell or gamma-radiation irradiation.Use wild-type K1735 cell (2 * 10 ⁶Cell/mouse) or Ag104 (3 * 10 ⁵Cell/mouse) immune stimulatory mouse.Measure the size that the perpendicular diameter of two maximums is assessed tumour by using calipers, and report average tumor area (mm ²) ± SD.The hair at the position of tumour subcutaneous transplantation shaved remove the measurement that helps tumour.

CD4 ⁺And/or CD8 ⁺The consumption in vivo of T lymphocyte and natural killer cell.T cell depletion (Chen as described herein, Ashe et al., Cell, 71,1093-102,1992), to mouse peritoneal injection CD4 monoclonal antibody (GK1.5, mouse IgG2b) or CD8 (169-4, mouse IgG 2a) or the mixing element of the two, 3 dosage are the 0.5mg/ mouse, inject continuously 3 days.Used every kind of monoclonal antibody 0.5mg in per subsequently 3 days, to keep this consumption.Inject the consumption that anti-asialo GM1 antibody is kept the NK cell by per 4 days abdominal cavities with the dosage of 30ul/ mouse.At the 12nd day, confirm the efficient that consumes with the splenocyte of every group of facs analysis.Subsequently, give the mouse subcutaneous transplanting tumour cell.

Enrichment procedure.With splenocyte in 96 holes flat culture plate (1 * 10 ⁵Individual cells/well) with 5 * 10 ⁵Individual from body, (3,000 rad) splenocyte (as antigen presenting cell) and tumour cell co-cultivation behind radiation exposure.After having hatched 72 hours, with in triplicate culture with 1 μ Ci ³The thymus pyrimidine pulse of [H] mark 16-18 hour (AmershamPharmacia, Biotech Piscataway, New Jersey), and measure intake.

In order to study which T cell at in-vitro multiplication, the method that provides according to the manufacturer (Molecular Probes, Eugene, Oregon), with splenocyte at the CFDASB of 2 μ M (5-(with-6)-Fluoresceincarboxylic acid diacetic acid amber acid ester) (den Haan, Lehar et al., J.Exp.Med., 192,1685-1695,2000) carried out hatching mark in, under existence of wild-type K1735 cell and non-existent situation, cultivated three days, and analyzed with FACS.

The cytotoxic lymphocyte activity test method.K1735-1D8 transplants back 2-4 week and puts to death mouse, and preparation splenocyte suspension.According to the rules, use anti-asialo GM1 antibody to add that (Cedarane, Ontario Canada) remove the NK cell to the rabbit complement, and (Costar Corp., Cambridge is Massachusetts) with 5 * 10 in one 24 well culture plate ⁶Individual splenocyte with 1 * 10 ⁵(12,000 rad) wild-type K1735 cell of-irradiation was cultivated 5 days.Use a kind of 4 hours ⁵¹The Cr method for releasing detects cell activity at different E/T ratios.

The ELISPOT method.Use mouse IFN-γ ELISPOT test kit (R﹠amp according to the method that the manufacturer provides; D Systems, Minneapolis, Minnesota), and by the flat-bed scanning device to flat board count (Cellular Technology Ltd., Cleveland, Ohio).

There is the active human T-cell of polyclone to increase and produces Th1 type lymphokine and make lysis.Figure 10 has shown the expression of anti-people CD3 scFv at Reh cell and T51 cell surface, and wherein, the former is the cell that a kind of reticuloendothelial cell is, the latter is a kind of cell of lymphocyte series.Show among the figure that the expression level of the anti-CD3 scFv of transfected cell gene product is very high.

By with the PBMC co-cultivation of taking from normal donor and detect and to learn, transfected Reh and T51 cell are compared with the ability of T51 cell with wild Reh, the amplification that the former can inducing T cell.Split the plain C of enzyme with silk and handle with transfected clone, increase in the training period to prevent them to wild-type.Transfected Reh that can express anti-CD3 scFv and T51 cell be with the amplification of dose-dependent pattern inducing T cell, and the Reh of wild-type and T51 cell then can (Figure 11).

Whether test to detect can stimulate the T cell from peripheral blood lymphocytes to kill transfected cell apace at the anti-CD3 scFv of tumor cell surface expression.Wild-type or transfected Reh and T51 cell are used respectively ⁵¹Cr carries out mark, carries out 8 hours with the peripheral blood lymphocytes of taking from normal donor ⁵¹The Cr release test.Figure 12 has shown that immobilized T cell kills transfected cell fast, but not wild-type cell, and causes dose-dependently ⁵¹The remarkable release of Cr.

The K1735-500A2 cell is had immunocompetently to repel from the body mouse.As seen in Figure 7, K1735-500A2 cell one transfected and that express anti-mouse CD3 scFv is crossed property ground and is grown in the mouse body, just be excluded subsequently.The cell growth of expressing CD80 is rapid, although than slow without cells transfected, this is consistent (Chen et al, J Exp Med, 179,523-532,1994) with the discovery that before had been in the news.

Have 10 mouse transplanted 3 times, the interval is 7 days respectively, uses 2 * 10 at every turn ⁶Individual anti-CD3 (500A2 scFv) transfectional cell is transplanted (not inoculating any tumour).9 control group mice are only injected phosphate buffered saline buffer (PBS).Then, two groups of mouse are all used 10 ⁶Individual wild-type K1735 cell stimulates.Wild-type K1735 cell forms tumour in all control group mice bodies, in 10 experimental mice, have in 6 mouse bodies not form tumour.Have 4 in the mice immunized body, to form tumour, but the time ratio that tumour forms is organized mouse evening without immunity (contrast).In any mouse body, toxicity or immunosuppressant evidence all do not occur, comprise the mouse of the tumour cell of the anti-CD3 scFv of duplicate injection transfection.

At the evidence that bystander effect K1735-500A2 cell and wild-type K1735 cytomixis formula occurred.In order to confirm to express the tumour cell of anti-CD3 scFv, for example, as the result of transfection in the body, whether can induce also can effectively anti-wild-type tumour cell immunne response, carried out two altogether with wild-type K1735 cell and the blended test mutually of K1735-500A2 cell.First test show, in the time of two types cell balanced mix, tumour can be degenerated through after the growth of short period of time in vivo.In second test is with 2 * 10 ⁶Individual wild-type K1735 cell and 2 * 10 ⁵Individual K1735-500A2 cell mixes mutually.Show that as Figure 13 compare during with independent transplanting, the growth of wild-type cell has been suppressed.

Cause tumor-selective T cell proliferation with K1735-500A2 cellular immunization.Collect the splenocyte of the mouse of the K1735-500A2 cell of refusing transplanting.Figure 14 shows the propagation of this splenocyte, splenocyte when external with through the wild-type K1735 of radiating irradiation cell co-cultivation the time, than splenocyte with through the antigenicity of radiating irradiation totally different from body sarcoma Ag104 cell co-cultivation the time degree of breeding bigger.Radiation exposure K1735-500A2 cellular immunization mouse boosting cell is unlike natural mouse boosting cell (contrast) molecular marker for increased proliferation.

Therefore, anti-CD3 scFv is at the quick killing tumor cell of the induced expression of tumor cell surface, and causes T cell proliferation.These characteristics have promoted the tumour-specific immunity, because the activation of the polyclone of the destruction of tumour cell and T cell produces tumour antigen, the latter is engulfed by sophisticated dendritic cell under the influence of the cytokine that produces at the T cell.The T cell by the anti-CD3 activation of polyclone at first by sensitization, then along with their identification by the tumour antigen of antigen presenting cell submission, tumour-specific T cell continues propagation.The plastidogenetic 1 type lymphokine of activated T, and T cell itself can be destroyed and look on tumour cell, and the transfection of tumour cell in vivo is described, express anti-CD3 scFv and may have result of treatment.

Embodiment 3 anti-4-1BB scFv are used for gene therapy for cancer

For make can stimulating immune system vaccine, be with existing technology (Hayden, Grosmaire et al., Tissue Antigens, 48,242-54,1996; Winberg, Grosmaireet al., Immunol Rev, 153,1996) one of structure can be encoded and be derived from hybridoma 1D8 (a kind of the anti-4-1 bb monoclonal antibody) (Melero, Shuford et al., Nat Med, 3,682-5,1997) the carrier of cell mating type scFv.To deriving from (Ward et al., J.Exp.Med., 170,1989) in the middle of the melanomatous cell of K1735, the immunogenicity of this cell is lower, and the expression level of I type major histocompatibility antigen is lower with this carrier transfection.Transfected cell can bring out the immune response of a kind of intensive Th1 type, CD4 ⁺, but not CD8 ⁺The T lymphocyte be essential to this reaction, natural killer cell has also participated in this reaction.Can repel wild-type K1735 tumour through the mouse of inoculation grows up to tubercle or grows in lung subcutaneous.The method of this invention can be resisted human patients effectively and the microtumor metastasis occur, comprise, for example those lower tumours of the expression level of I type major histocompatibility antigen.

Mouse and tumor cell line.Bought the female C3H/HeN mouse (Taconic, Germantown, New York) in 6 to 8 ages in week.K1735 is the malignant melanoma that a kind of C3H/HeN takes place, and has selected a kind of stable clone M2 (Fidler and Hart, CancerRes, 41,3266-3267,1981).Consistent with previous discovery, find its MHC I class expression very low (data not shown).Ag104 (Ward, Koeppen et al., J.Exp.Med., 170,1989) is long in C3H/HeN mouse a kind of spontaneous fibrosarcoma on one's body.YAC-1 available from American Type Culture Collection (Rockville, Maryland).Animal facility be through ALAC authentication and experimental technique approve through corresponding public animal association.

Antibody.R-phycoerythrin (PE)-coupling monoclonal antibody GK1.5 (anti-mouse CD4), AF3-12.1 (the anti-H-2K of 53-6.7 (anti-mouse CD8a) and purifying ^K) available from Pharmingen company (San Diego, California), R-PE link coupled goat F (ab ') ₂Anti-human IgG available from Biosource International company (Camarillo, California).Monoclonal antibody 169-4 (anti-CD8) from doctor R.Mittler (Emory University, Atlanta, Georgia).GK1.5 (anti-CD4) produces from ATCC hybrid cell knurl.The anti-asialo GM of rabbit antibody available from Wako purified reagent factory (Richmond, Virginia), the rabbit igg of purifying available from Sigma and Rockland company (Gilbertsville, Pennsylvania).

The transfection of carrier and K1735 cell.Described the variable region clone, scFv makes up and scFv expresses method (Gilliland, Norris et al., Tissue Antigens, 47,1-20,1996 that produce; Hayden et al., Tissue Antigens, 48,242-54,1996; Winberg, Grosmaire et al., Immunol Rev, 153,1996).The present invention adopts the gene from anti-CD3 hybridoma 500A2 or anti-4-lBB hybridoma 1D8 variable region to obtain the expression (Hayden of cell in conjunction with the cell surface of 500A2 scFv or 1D8 scFv, Grosmaire etal..Tissue Antigens, 48,242-54,1996; Winberg, Grosmaire et al., Immunol Rev, 153,1996).In order to express scFv, used membrane spaning domain and tenuigenin tail from CD80 because its mediated cytoskeleton adhere to cell and cells contacting the time crosslinked (Doty and dark, J Immunol, 157,3270-9,1996; Doty andClark, J Immunol, 161,2700-7,1998).The scFv gene integration in pLNCX, is passed through CaPO ₄Intermediate processing transfection RetroPack ^TMThe PT67 packing cell (ClontechLaboratories.Inc, Palo Alto, California).Use is from the substratum transfection wild-type K1735 cell of these cells.Use the goat anti-human igg of PE mark that the G418 resistance clone is carried out mark, be used for the expression of scFv at cell surface.

Zooscopy.Mouse, 5 or 10/group, back one side subcutaneous transplantation 2 * 10 ⁶Individual wild-type K1735 cell or K1735-500A2 cell or (12,000 rad) the wild-type K1735 cell that shines through gamma-radiation.Use wild-type K1735 cell (2 * 10 ⁶Cell/mouse) or Ag104 (3 * 10 ⁵Cell/mouse) immune stimulatory mouse.Measure the size that the perpendicular diameter of two maximums is assessed tumour by using calipers, and report average tumor area (mm ²) ± SD.The hair at the position of tumour subcutaneous transplantation shaved remove, be beneficial to the measurement of tumour.

In first test, give mouse in tail lateral vein intravenous injection 3 * 10 ⁵Wild-type K1735 cell is to cause lung's metastasis (Kahn et al., J Immunol, 146,3235-3241,1991).After three days,, transplant once co-transplantation four times weekly at a side subcutaneous transplantation K1735-1D8 cell of mouse back.After the transplanting wild-type cell 37 days, mouse is put to death.Give the Indian ink of mouse intratracheal injection (concentration with 15% is dissolved in the phosphate buffered saline buffer), lungs are taken out, uncoloured metastasis sets off visible (the Estin et al. of naked eyes down in the healthy tissues of black, Proc Nati Acad Sci USA, 85,1052-6,1988).

CD4 ⁺And/or CD8 ⁺The consumption in vivo of T lymphocyte and natural killer cell.T cell depletion (Chen, Ashe et al., Cell, 71 as described herein, 1093-102,1992), to mouse peritoneal injection CD4 monoclonal antibody (GK1.5, mouse IgG2b) or CD8 (169-4, mouse IgG 2a) or the mixing element of the two 3 times, with the injection 3 days continuously of 0.5mg/ mouse.Used every kind of monoclonal antibody 0.5mg in per subsequently 3 days, to keep this consumption.Inject the consumption that anti-asialo GM1 antibody is kept the NK cell by per 4 days abdominal cavities with the dosage of 30ul/ mouse.At the 12nd day, confirm the efficient that consumes with the splenocyte of every group of facs analysis.Subsequently, give the mouse subcutaneous transplanting tumour cell.

Enrichment procedure.With splenocyte in 96 holes flat culture plate (1 * 10 ⁵Individual cells/well) with 5 * 10 ⁵Individual from body, (3,000 rad) splenocyte (as antigen presenting cell) and tumour cell co-cultivation behind radiation exposure.After having hatched 72 hours, with in triplicate culture with 1 μ Ci ³The thymus pyrimidine pulse of [H] mark 16-18 hour (AmershamPharmacia, Biotech Piscataway, New Jersey), and measure intake.

In order to study which T cell at in-vitro multiplication, the method that provides according to the manufacturer (Molecular Probes, Eugene, Oregon), with splenocyte at the CFDASB of 2 μ M (5-(with-6)-Fluoresceincarboxylic acid diacetic acid amber acid ester) (den Haan, Lehar et al., J.Exp.Med., 192,1685-1695,2000) carried out hatching mark in, under existence of wild-type K1735 cell and non-existent situation, cultivated three days, and analyzed with FACS.

The cytotoxic lymphocyte activity test method.K1735-1D8 transplants back 2-4 week and puts to death mouse, and preparation splenocyte suspension.According to the rules, use anti-asialo GM1 antibody to add that (Cedarane, Ontario Canada) remove the NK cell to the rabbit complement, and (Costar Corp., Cambridge is Massachusetts) with 5 * 10 in one 24 well culture plate ⁶Individual splenocyte with 1 * 10 ⁵(12,000 rad) wild-type K1735 cell of-irradiation was cultivated 5 days.Use a kind of 4 hours ⁵¹The Cr method for releasing detects cell activity at different E/T ratios.

The ELISPOT method.Use mouse IFN-γ ELISPOT test kit (R﹠amp according to the method that the manufacturer provides; D Systems, Minneapolis, Minnesota), and by the flat-bed scanning device to flat board count (Cellular Technology Ltd., Cleveland, Ohio).

Immunohistochemical methods.To organize taking-up in 10-30 days after the injection tumour, use 10% formalin fixed, the section of 4-6 μ m is made in sealing, and with Vector ABC test kit (California, Berlin lid nurse, Vector laboratory) dyeing, detects CD4 according to operational manual again ⁺And CD8 ⁺The T cell.This part operation also can be adopted H-E dyeing.

By needs CD4 ⁺The mechanism that T cell and natural killer cell participate in is repelled the K1735-1D8 cell.The anti-4-1BB scFv of cell bonded is cloned into (Figure 15 a) among the reverse transcription carrier pLNCX.With the cell (Fidlerand Hart, Cancer Res, 41,3266-3267,1981) (be called as wild-type K1735 cell) of this construct transfection to the transfer M2 clone who takes from K1735.Transfected clone, promptly the K1735-1D8 cell is understood at the high-caliber anti-4-1BB scFv of its surface expression (Figure 15 b).

Wild-type K1735 cell is transplanted to natural at subcutaneous (s.c.) and is grown rapidly behind body (C3H) mouse.Form area when the beginning with the K1735-1D8 cell of above-mentioned subcutaneous transplantation same dose and be about 30mm although use ²Tumour, but behind subcutaneous transplantation the 20th day the time, these tumor regressions and disappeared (Figure 15 c).With what in kind make up, the control vector transfection wild-type K1735 cell of the anti-people CD28scFv that encodes, the cell after the transfection is identical with wild-type K1735 cell in the intravital growth velocity of C3H mouse.

In order to study CD4 ⁺And CD8 ⁺T lymphocyte and the effect of natural killer cell in K1735-1D8 cell process of extinction, give in the natural mouse peritoneal injection monoclonal antibody to remove CD8 ⁺, CD4 ⁺, or CD4 ⁺And CD8 ⁺The T cell, or intraperitoneal injection anti--asialo GM1 rabbit antibody, to remove natural killer cell.To mice in control group injection rat IgG.After 12 days, the splenocyte of taking from same mouse being carried out when facs analysis shows that a large amount of target cells have exhausted, to the equal subcutaneous transplantation K1735-1D8 of the mouse of each group cell.By anti-CD8 monoclonal antibody of injection or control rats IgG, removed CD4 ⁺The T cell is or/and CD8 ⁺In the mouse body of T cell, the growth kinetics of K1735-1D8 cell is identical, and this makes that the upgrowth situation of K1735-1D8 cell is identical with wild-type K1735 cell.Cell can be grown in the depleted mouse body of all natural killer cells, although the velocity ratio of this growth is wanted slow a lot (Figure 15 d) in the intravital speed of the depleted mouse of CD4.

Carry out immunity with the K1735-1D8 cell, the memory of utilization immunity and specificity are induced the immunity at wild-type K1735 cell.C3H mouse is divided into 10 every group, every twice K1735-1D8 cell of 10 days subcutaneous transplantations or through the wild-type K1735 of radiation exposure cell; Give mice in control group subcutaneous injection phosphate buffered saline buffer (PBS).After 10 days, give the injected in mice wild-type cell.Through K1735-1D8 cellular immunization but do not accept mouse through the wild-type K1735 of radiation exposure cellular immunization and suppressed wild-type cell (Figure 16 a).Be enough to protect the wild-type K1735 cell of body antagonism transplanting separately with K1735-1D8 cellular immunization.

Suppress wild-type cell after two months, the mouse that can repel the K1735-1D8 cell through immunity can repel once more that (Figure 16 a) by the wild-type cell of injection transplantation.In contrast be, from the sarcoma Ag104 cell of no antigen dependency in " repulsion type " mouse body with the same good (Figure 16 b) that in natural contrast body, grow.

In immunity through twice antagonism K1735-1D8, and after this can repel in the mouse of transplanted wild-type cell, have approximately 20% can be longer than 4 months with occurring skin fade (Figure 16 c) during examining.There are not other autoimmune signs.

The K1735-1D8 cell is effective as a kind of treatment vaccine.Carry out three and given the test of transplanting the K1735-1D8 cell in the mouse body that has formed wild-type K1735 tumour.First is to be about 30mm at existing surface-area ²The test of doing in the mouse body of Subcutaneous tumor.When giving wherein one group of first week around every at the back, the wild-type tumour to side injection K1735-1D8 cell.To the wild-type K1735 cell of another group transplanting, give the 3rd group of subcutaneous injection phosphate buffered saline buffer (PBS) through irradiation.In the mouse body of the useful wild-type K1735 cellular immunization of all mice in control group and institute, all formed the wild-type tumour through shining, in contrast be, in by the mouse of immune anti-K1735-1D8 cell, four intravital tumor regressions of mouse in five mouse (Figure 17 a), when test stops after three months, these four mouse still do not have tumour, do not have the sign of poisoning yet.The 5th the nodular size of mouse tumor also reduced, as long as this mouse is accepted the K1735-1D8 Transplanted cells.

In second test, be to begin at 4 or 8 days with preceding 1 day of wild-type K1735 Transplanted cells or after transplanting, give mouse subcutaneous injection K1735-1D8 cell weekly.Injection 1D8 monoclonal antibody is with making comparisons in identical medication time is given the mouse peritoneal of other groups.Give control group intraperitoneal injection phosphate buffered saline buffer (PBS).As shown in the table 4, must after accepting the wild-type cell transplanting, put to death all mice in control group in 49 days, because occurred in their bodies 〉=100mm ²Tumour.In contrast be, begin with the K1735-1D8 cell inoculation or give the mouse of 1D8 monoclonal antibody in the day before yesterday of transplanting wild-type cell, after the transplanting wild-type cell, do not have tumour in the mouse body during off-test in 70 days.Beginning in the 4th or the 8th day after transplanting with wild-type cell by the mouse of immune anti-K1735-1D8, does not have the tumour that can detect during preceding 28 days of observing, but after vaccinate no longer, 4 in these 10 mouse tumour occurred.The corresponding K1735-1D8 group of the tumorigenic time ratio of the mouse mouse of 8 days injection monoclonal antibodies is wanted early after the WT cell formation tumour, but the survival rate of two groups of mouse is as broad as long.Be divided into several sections with transplanting the tumour of gathering in the crops in 20 days behind the wild-type cell, and carry out H﹠amp; E dyeing and immunohistochemical methods assessment.A tumor nodule of taking from phosphate buffered saline buffer (PBS) control group mice includes many tumour cells and a spot of CD4 ⁺And CD8 ⁺The T lymphocyte, and the mouse that butt joint receptor 3 monoclonal antibody was injected the 8th day carries out same analysis.Corresponding therewith is in taking from the tumor nodule that carries out the 8th day mouse of anti-K1735-1D8 immunity first, to contain a large amount of CD4 ⁺And CD8 ⁺The T lymphocyte, and only contain the tumour cell (Figure 17 b) of minute quantity.

The 3rd test also is five mouse/groups, and we give mouse mainline 3 * 10 in this test ⁵Individual wild-type K1735 cell shifts to form lung.After three days, subcutaneous transplantation K1735-1D8 cell continues one month altogether weekly; Give mice in control group intravenous injection phosphate buffered saline buffer (PBS).A control group mice death is being arranged, or test is ended after accepting wild-type K1735 cell 37 days.At that time, the lungs of each control group mice all have 500 metastasis of surpassing, and under comparing, such metastasis is less than 10 (Figure 17 c) in the mice immunized lungs.

Can bring out Th1 type immunne response with K1735-1D8 cellular immunization.Picked-up by tritium-labeled thymus pyrimidine is measured, the propagation degree through the splenocyte of the anti-K1735-1D8 mouse of immunity taken from be approximately take from natural mouse or through the twice of the splenocyte of the mouse of the wild-type K1735 of radiation exposure cellular immunization (Figure 18 a).Take from the splenocyte of K1735-1D8 mice immunized with after the wild-type K1735 of radiation exposure cell co-cultivation, can increase about 4 times, then can not with Ag104 cell co-cultivation.Also can be to taking from natural mouse and carrying out proliferation experiment through the splenocyte of the mouse of the anti-K1735-1D8 of immunity, splenocyte with or not with hatch jointly through the wild-type K1735 of radiation exposure cell before with CFDA SE (den Haan, Lehar et al., J.Exp.Med., 192,1685-1695,2000) carry out mark.Take from the CD4 of K1735-1D8 immune mouse ⁺And CD8 ⁺Splenocyte amplification swift and violent (Figure 18 b), intensive amplifies in the present wild-type K1735 cell.Amplification does not appear in the splenocyte of taking from natural mouse.

The splenocyte of immune anti-K1735-1D8 mouse of hanging oneself recently becomes to carry the K1735-WT tumour from natural mouse, and (splenocyte of Figure 19 mouse a), what produce IFN r in ELISPOT analyzes wants many.In the test of table 4, the ELISPOT that carries out with splenocyte analyzes the day before yesterday or four days afterwards that has confirmed transplanting wild-type K1735, with the K1735-1D8 mice immunized be respond active, in the ban with wild-type K1735 cell co-cultivation reactive behavior higher (Figure 19 b) in the time of 3 days.Strong reaction activity appears at the group of wild-type cell immunity the day before yesterday, and this may be because tumor load is less.Mouse that the anti-4-1BB monoclonal antibody of using by oneself is injected or the splenocyte of natural mouse do not show reactive behavior.

To take from by the splenocyte of the mouse of the anti-K1735-1D8 of immunity and through the wild-type K1735 cell of irradiation and hatch jointly 5 days, use 4 hours then ⁵¹The Cr release experiment is analyzed.Do not remove natural killer cell, K1735, Ag104 and YAC cell can cleaved with amount much at one (Figure 19 c) in advance.Yet, hatch the removal natural killer cell jointly if earlier splenocyte and the anti-asialo GM1 of rabbit antibody are added complement, compare with Ag104 or YAC cell so, it can show the cytotoxic lymphocyte activity of antagonism wild-type K1735 cell significantly, although this activity is lower, this activity can also be suppressed (Figure 19 d) by the monoclonal antibody of anti-I class major histocompatibility antigen.

The evidence that bystander effect when K1735-1D8 cell and wild-type K1735 cytomixis formula, occurs.In order to study the tumour cell of expressing anti-4-1BB scFv in vivo, for example as the result of transfection in a kind of body, whether can bring out a kind of immunne response, this effect that also has antagonism wild-type tumour cell of replying has now been carried out two with the test of studying after wild-type K1735 cell and the K1735-1D8 cytomixis.First test shown after two kinds of cell balanced mix, just taken place to disappear after the of short duration in vivo growth of tumour.In second test, with 2 * 10 ⁶Individual wild-type K1735 cell and 2 * 10 ⁵Individual K1735-1D8 cell mixes mutually.Show that as Figure 20 compare with their independent situations when transplanted, the growth of wild-type cell has been suppressed.

Therefore, be derived from the anti-4-1 bb hybridoma 1D8, can express cell can be repelled CD from the body mouse in conjunction with the K1735-1D8 cell of scFv ⁴+ T lymphocyte, natural killer cell, but not CD8 ⁺The T lymphocyte all be the rejection necessary.In addition, the immunity of anti-K1735-1D8 can be brought out and have a reaction of immunological memory and specific systemic immunity at wild-type K1735.In contrast, use mouse can not show provide protection repeatedly to the invasion and attack of wild-type cell through the wild-type K1735 of radiation exposure cellular immunization.This with show that K1735 is in transfection and the data of low immunogenicity are still arranged after expressing CD80 is consistent.Analyzed in vitro shows, can be resisted the K1735-1D8 cell by the splenocyte of immune mouse.Carry the mouse inoculation vaccine no matter when tumor growth is in subcutaneous or lung, all be to have curative effect to tumour.

This low immunogenicity of viewed antagonism wild-type K1735, the tumor treatment effect prompting that I type major histocompatibility antigen expression amount is extremely low, with the tumour cell transfection, make it express anti-4-1BB scFv, then as use from body or allogeneic vaccine can destroy cancer patient to accept traditional treatment after remaining micro metastasis.

ScFv is from hybridoma 5B9 for people 4-1BB specificity, the course of processing and above-described G19-4, and the course of processing of 500A2 and 1D8 scFv is consistent.Figure 21 has shown that 5B9 scFv sequence merges mutually with membrane spaning domain and the endochylema tail of human IgG1's hinge, CH2 and CH3 structural domain and people CD80.

***

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Ranga，U.，C.Woffendin，et?al.(1998).“Enhanced?T?cell?engraftment?after?retroviraldelivery?of?an?antiviral?gene?in?HIV-infected?individuals.” Proc.Natl .Acad.Sci.95：1201-1206.

Rodolfo，M.，C.Zilocchi，et?al.(1999).“IL-4-transduced?tumor?cell?vaccine?inducesimmunoregulatory?type?2?CD8T?lymphocytes?that?cure?lung?metastases?upon?adoptive?transfer.” J?I mmunol?163(4)：1923-8.

Rosenberg，S.A.(1995).Cell?transfer?therapy：clinical?applications. Biologic?Therapy?of Cancer(Chapter?19).V.T.DeVita，S.Hellman?and?S.A.Rosenberg.Philadelphia，PA，J.P.Lippincott：487.

Rosenberg，S.A.，J.C.Yang，et?al.(1998).“Immunologic?and?therapeutic?evaluation?of?asynthetic?peptide?vaccine?for?the?treatment?of?patients?with?metastatic?melanoma[seecomments].” Nat?Med?4(3)：321-7.

Schoenberger，S.P.，L.E.Jonges，et?al.(1998).“Efficient?direct?priming?of?tumor-specific?cytotoxic?T?lymphocyte?in?vivo?by?an?engineered?APC.” Cancer?Res?58(14)：3094-100.

Schwartz，R.H.(1989).“Acquisition?of?immunologic?self-tolerance.” Cell?57(7)：1073-81.

Shiraki，K.，N.Tsuji，et?al.(1997).“Expression?of?Fas?ligand?in?liver?metastases?ofhuman?colonic?adenocarcinomas.” Proc?Natl?Acad?Sci?USA?94(12)：6420-5.

Shuford，W.W.，K.Klussman，et?al.(1997).“4-1BB?costimulatory?signals?preferentiallyinduce?CD8+T?cell?proliferation?and?lead?to?the?amplification?in?vivo?of?cytotoxic?T?cellresponses.” J?Exp?Med?186(1)：47-55.

Sulitzeanu，D.(1993).“Immunosuppressive?factors?in?human?cancer.” Adv?Cancer?Res60：247-267.

Takahashi，C.，R.S.Mittler，et?al.(1999).“Cutting?edge：4-1BB?is?a?bona?fide?CD8?T?cellsurvival?signal.” J?Immunol?162(9)：5037-40.

Thompson，C.B.，T.Lindsten，et?al.(1989).“CD28?activation?pathway?regulates?theproduction?of?multiple?T-cell-derived?lymphokines/cytokines.” Proc?Natl?Acad?Sci?U?S?A?86(4)：1333-7.

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Walunas，T.L.，C.Y.Bakker，et?al.(1996).“CTLA-4?ligation?blocks?CD28-dependent?Tcell?activation.” J?Exp?Med?183(6)：2541-50.

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***

All patents of quoting herein and mentioning, publication, scientific and technical article, website and other reference and data all are the embodiments of one of ordinary skill in the art's level of the present invention, all complete separately quoting as a reference.The applicant keep with arbitrarily or all from any this patent, publication, scientific and technical article, webpage, whole rights of adding this part patent specification to of available electronic information and other reference materials or document.

Special method and composition described herein is represented embodiment preferred, is exemplary, and its purpose does not lie in scope of the present invention is limited.Aspect other that on basis of the present invention, associate and embodiment be also contained in claims of the present invention institute restricted portion.For those skilled in the art, under the prerequisite that does not deviate from the scope and spirit of the present invention, it being done various substituting and correction, is very conspicuous.In this this invention as illustrations, if lack any integral part or a plurality of integral part, limit or a plurality of limit when this specially illustrates, still can be implemented.Therefore, in any situation of this explanation, for example in embodiments of the invention or the embodiment, any term " comprises ", " substantially by ... form ", " comprising " and " by ... form " can be illustrated in the book any two other term alternative.Simultaneously, " comprising ", " comprising ", can extend implication and unrestricted of " containing " or the like term.Be with different sequence of steps with step in this method of describing explanation and still set up now, be not limited to herein or specified sequence of steps in the claim.Under any circumstance, all this patent can not be limited in embodiment and the embodiment scope of disclosure.This patent never can be interpreted as being subjected to the restriction of the report that any examiner or any other official and patent and trade mark office staff work out, unless this report is special, does not add restriction or the written reaction of applicant and clearly adopts reservation.

Term that adopts and expression are the terms that is used for explaining; it is not limitation of the present invention; not by using such term and representation to get rid of any synonymous expression intention of indicating characteristic and description or certain part herein, but be to be appreciated that various variations may be within the scope of protection of present invention.Therefore, although be appreciated that present invention is open by embodiment preferred and optional characteristic, those skilled in the art can make this and changing and adjustment, and this modification and variation are in the scope of dependent claims definition of the present invention.

The present invention this carried out widely, general description.Fall into the general openly narrower kind and the subgenus of scope and also constitute a part of the present invention.In general announcement the each limits kind and inferior general grouping has also constituted part of the present invention.This comprises describes the generality of invention, and subsidiary restrictive clause or negative restriction are to remove any material, and do not consider whether the material of removing has carried out special description at this.

Other embodiment all is included in the middle of the following claim.In addition, characteristics of the present invention or aspect are described in the mode of Ma Kushi (Markush) group, and those skilled in the art are to be appreciated that the present invention also can be described by the member of Ma Kushi group or the mode of member's subgroup.

Sequence table

<110>LEDBETTER，JEFFREY

HAYDEN-LEDBETTER，MARTHA

HELLSTROM，INGEGERD

HELLSTROM，KARL?ERIK

＜120〉identification CD3 or the antibody of 4-1BB or the tumor response lymphocyte activation of gene mediated

<130>034474.0004

<140>10/107,991

<141>2002-04-26

<150>60/286,585

<151>2001-04-26

<160>5

＜170〉patent version 2 .1

<210>1

<211>1687

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: mouse-people's hybrid gene

<220>

<221>CDS

<222>(7)(1671)

<220>

＜221〉signal peptide

<222>(7)(75)

＜223〉the coding both sides are connected with the L6 V κ leading peptide of Hind III and SalI restriction enzyme site

<220>

＜221〉V_ district

<222>(76)(406)

＜223〉coding Gl9-4 mouse anti human CD3 VL structural domain

<220>

＜221〉misc_ feature

<222>(406).(451)

＜223〉coding (Gly4Ser) 3 joints

<220>

＜221〉V_ district

<222>(451).(816)

＜223〉G19-4 mouse anti human CD3 VH structural domain

<220>

＜221〉misc_ feature

<222>(816)(1521)

＜223〉coding human IgG1 Fc structural domain (hinge area, CH2, CH3)

<220>

＜221〉misc_ feature

<222>(1521)..(1687)

＜223〉coding people CD80 strides film and tenuigenin structural domain and the restriction enzyme site that is connected

<400>1

aagctt?atg?gat?ttt?caa?gtg?cag?att?ttc?agc?ttc?ctg?cta?atc?agt 48

Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser

1 5 10

gct?tca?gtc?ata?atg?tcc?aga?gga?gtc?gac?atc?cag?atg?aca?cag?act 96

Ala?Ser?Val?Ile?Met?Ser?Arg?Gly?Val?Asp?Ile?Gln?Met?Thr?Gln?Thr

15 20 25 30

aca?tcc?tcc?ctg?tct?gcc?tct?ctg?gga?gac?aga?gtc?acc?atc?agt?tgc 144

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

35 40 45

agg?gca?agt?cag?gac?att?cgc?aat?tat?tta?aac?tgg?tat?cag?cag?aaa 192

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

50 55 60

cca?gat?gga?act?gtt?aaa?ctc?ctg?atc?tac?tac?aca?tca?aga?tta?cac 240

Pro?Asp?Gly?Thr?Val?Lys?Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Arg?Leu?His

65 70 75

tca?gga?gtc?cca?tca?agg?ttc?agt?ggc?agt?ggg?tct?gga?aca?gat?tat 288

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

80 85 90

tct?ctc?acc?att?gcc?aac?ctg?caa?cca?gaa?gat?att?gcc?act?tac?ttt 336

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

95 100 105 110

tgc?caa?cag?ggt?aat?acg?ctt?ccg?tgg?acg?ttc?ggt?gga?ggc?acc?aaa 384

Cys?Gln?Gln?Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys

115 120 125

ctg?gta?acc?aaa?cgg?gag?ctc?ggt?ggc?ggt?ggc?tcg?ggc?ggt?ggt?ggg 432

Leu?Val?Thr?Lys?Arg?Glu?Leu?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly

130 135 140

tcg?ggt?ggc?ggc?gga?tct?atc?gat?gag?gtc?cag?ctg?caa?cag?tct?gga 480

Ser?Gly?Gly?Gly?Gly?Ser?Ile?Asp?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly

145 150 155

cct?gaa?ctg?gtg?aag?cct?gga?gct?tca?atg?tcc?tgc?aag?gcc?tct?ggt 528

Pro?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Met?Ser?Cys?Lys?Ala?Ser?Gly

160 165 170

tac?tca?ttc?act?ggc?tac?atc?gtg?aac?tgg?ctg?aag?cag?agc?cat?gga 576

Tyr?Ser?Phe?Thr?Gly?Tyr?Ile?Val?Asn?Trp?Leu?Lys?Gln?Ser?His?Gly

175 180 185 190

aag?aac?ctt?gag?tgg?att?gga?ctt?att?aat?cca?tac?aaa?ggt?ctt?act 624

Lys?Asn?Leu?Glu?Trp?Ile?Gly?Leu?Ile?Asn?Pro?Tyr?Lys?Gly?Leu?Thr

195 200 205

acc?tac?aac?cag?aaa?ttc?aag?ggc?aag?gcc?aca?tta?act?gta?gac?aag 672

Thr?Tyr?Asn?Gln?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys

210 215 220

tca?tcc?agc?aca?gcc?tac?atg?gag?ctc?ctc?agt?ctg?aca?tct?gaa?gac 720

Ser?Ser?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Leu?Ser?Leu?Thr?ser?Glu?Asp

225 230 235

tct?gca?gtc?tat?tac?tgt?gca?aga?tct?ggg?tac?tat?ggt?gac?tcg?gac 768

Ser?Ala?Val?Tyr?TyL?Cys?Ala?Arg?Ser?Gly?Tyr?Tyr?Gly?Asp?Ser?Asp

240 245 250

tgg?tac?ttc?gat?gtc?tgg?ggc?gca?ggg?acc?acg?gtc?acc?gtc?tcc?tct 816

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

255 260 265 270

gat?ctg?gag?ccc?aaa?tct?tct?gac?aaa?act?cac?aca?agc?cca?ccg?agc 864

Asp?Leu?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Ser?Pro?Pro?Ser

275 280 285

cca?gca?cct?gaa?ctc?ctg?ggg?gga?tcg?tca?gtc?ttc?ctc?ttc?ccc?cca 912

Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Ser?Ser?Val?Phe?Leu?Phe?Pro?Pro

290 295 300

aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc?aca?tgc 960

Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys

305 310 315

gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag?ttc?aac?tgg 1008

Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp

320 325 330

tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg?cgg?gag 1056

Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu

335 340 345 350

gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc?gtc?ctg 1104

Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

355 360 365

cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc?tcc?aac 1152

His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn

370 375 380

aaa?gcc?ctc?cca?gcc?ccc?atc?gag?aaa?acc?atc?tcc?aaa?gcc?aaa?ggg 1200

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

385 390 395

cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc?cgg?gat?gag 1248

Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu

400 405 410

ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc?ttc?tat 1296

Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr

415 420 425 430

ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg?gag?aac 1344

Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

435 440 445

aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc?ttc?ttc 1392

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe

450 455 460

ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag?cag?ggg?aac 1440

Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn

465 470 475

gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac?cac?tac?acg 1488

Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr

480 485 490

cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?gcg?gat?cct?tcg?aac?ctg 1536

Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Ala?Asp?Pro?Ser?Asn?Leu

495 500 505 510

ctc?cca?tcc?tgg?gcc?att?acc?tta?atc?tca?gta?aat?gga?att?ttt?gtg 1584

Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile?Phe?Val

515 520 525

ata?tgc?tgc?ctg?acc?tac?tgc?ttt?gcc?cca?aga?tgc?aga?gag?aga?agg 1632

Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu?Arg?Arg

530 535 540

agg?aat?gag?aga?ttg?aga?agg?gaa?agt?gta?cgc?cct?gta?taaatcgata 1681

Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

545 550 555

ctcgag 1687

<210>2

<211>1696

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: mouse-people's hybrid gene

<220>

<221>CDS

<222>(13)..(1680)

<220>

＜221〉signal peptide

<222>(13)..(71)

＜223〉coding 5B9 mouse anti human 4-1BB VL leader peptide sequences

<220>

＜221〉V_ district

<222>(72)..(412)

＜223〉coding 5B9 mouse anti human 4-1BB VL structural domain

<220>

＜221〉misc_ feature

<222>(413)..(459)

＜223〉coding (Gly4Ser) 3 joint polypeptide

<220>

＜221〉V_ district

<222>(460)..(826)

＜223〉coding 5B9 mouse anti human 4-1BB VII structural domain

<220>

＜221〉misc_ feature

<222>(827)..(1527)

＜223〉coding human IgG1 Fc structural domain (hinge area, CH2, CH3)

<220>

＜221〉misc_ feature

<222>(1528)..(1696)

＜223〉the coding people CD80 that has a restriction site strides film and tenuigenin structural domain

<400>2

aagcttgccg?cc?atg?agg?ttc?tct?gct?cag?ctt?ctg?ggg?ctg?ctt?gtg?ctc 51

Met?Arg?Phe?Ser?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Val?Leu

1 5 10

tgg?atc?cct?gga?tcc?act?gca?gat?att?gtg?atg?acg?cag?gct?gca?ttc 99

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

15 20 25

tcc?aat?cca?gtc?act?ctt?gga?aca?tca?gct?tcc?atc?tcc?tgc?agg?tct 147

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

30 35 40 45

agt?aag?agt?ctc?cta?cat?agt?aat?ggc?atc?act?tat?ttg?tat?tgg?tat 195

Ser?Lys?Ser?Leu?Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr

50 55 60

ctg?cag?aag?cca?ggc?cag?tct?cct?cag?ctc?ctg?att?tat?cag?atg?tcc 243

Leu?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Gln?Met?Ser

65 70 75

aac?ctt?gcc?tca?gga?gtc?cca?gac?agg?ttc?agt?agc?agt?ggg?tca?gga 291

Asn?Leu?Ala?Ser?Gly?Val?Pro?Asp?Arg?Phe?Ser?Ser?Ser?Gly?Ser?Gly

80 85 90

act?gat?ttc?aca?ctg?aga?atc?agc?aga?gtg?gag?gct?gag?gat?gtg?ggt 339

Thr?Asp?Phe?Thr?Leu?Arg?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly

95 100 105

gtt?tat?tac?tgt?gct?caa?aat?cta?gaa?ctt?ccg?ctc?acg?ttc?ggt?gct 387

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

110 115 120 125

ggg?acc?aag?ctg?gag?ctg?aaa?cgg?ggt?ggc?ggt?ggc?tcg?ggc?ggt?ggt 435

Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly

130 135 140

ggg?tcg?ggt?ggc?ggc?gga?tcg?tca?cag?gtg?cag?ctg?aag?cag?tca?gga 483

Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ser?Gln?Val?Gln?Leu?Lys?Gln?Ser?Gly

145 150 155

cct?ggc?cta?gtg?cag?tcc?tca?cag?agc?ctg?tcc?atc?acc?tgc?aca?gtc 531

Pro?Gly?Leu?Val?Gln?Ser?Ser?Gln?Ser?Leu?Ser?Ile?Thr?Cys?Thr?Val

160 165 170

tct?ggt?ttc?tca?tta?act?acc?tat?gct?gta?cac?tgg?gtt?cgc?cag?tct 579

Ser?Gly?Phe?Ser?Leu?Thr?Thr?Tyr?Ala?Val?His?Trp?Val?Arg?Gln?Ser

175 180 185

cca?gga?aag?ggt?ctg?gag?tgg?ctg?gga?gtg?ata?tgg?agt?ggt?gga?atc 627

Pro?Gly?Lys?Gly?Leu?Glu?Trp?Leu?Gly?Val?Ile?Trp?Ser?Gly?Gly?Ile

190 195 200 205

aca?gac?tat?aat?gca?gct?ttc?ata?tcc?aga?ctg?agc?atc?acc?aag?gac 675

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

210 215 220

gat?tcc?aag?agc?caa?gtt?ttc?ttt?aaa?atg?aac?agt?ctg?caa?cct?aat 723

Asp?Ser?Lys?Ser?Gln?Val?Phe?Phe?Lys?Met?Asn?Ser?Leu?Gln?Pro?Asn

225 230 235

gac?aca?gcc?att?tat?tac?tgt?gcc?aga?aat?ggg?ggt?gat?aac?tac?cct 771

Asp?Thr?Ala?Ile?Tyr?Tyr?Cys?Ala?Arg?Asn?Gly?Gly?Asp?Asn?Tyr?Pro

240 245 250

tat?tac?tat?gct?atg?gac?tac?tgg?ggt?caa?gga?acc?tca?gtc?acc?gtc 819

Tyr?Tyr?Tyr?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr?Val

255 260 265

tcc?tct?gat?ctg?gag?ccc?aaa?tct?tct?gac?aaa?act?cac?aca?agc?cca 867

Ser?Ser?Asp?Leu?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Ser?Pro

270 275 280 285

ccg?agc?cca?gca?cct?gaa?ctc?ctg?ggg?gga?tcg?tca?gtc?ttc?ctc?ttc 915

Pro?Ser?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Ser?Ser?Val?Phe?Leu?Phe

290 295 300

ccc?cca?aaa?ccc?aag?gac?acc?ctc?atg?atc?tcc?cgg?acc?cct?gag?gtc 963

Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val

305 310 315

aca?tgc?gtg?gtg?gtg?gac?gtg?agc?cac?gaa?gac?cct?gag?gtc?aag?ttc 1011

Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe

320 325 330

aac?tgg?tac?gtg?gac?ggc?gtg?gag?gtg?cat?aat?gcc?aag?aca?aag?ccg 1059

Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro

335 340 345

cgg?gag?gag?cag?tac?aac?agc?acg?tac?cgt?gtg?gtc?agc?gtc?ctc?acc 1107

Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr

350 355 360 365

gtc?ctg?cac?cag?gac?tgg?ctg?aat?ggc?aag?gag?tac?aag?tgc?aag?gtc 1155

Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val

370 375 380

tcc?aac?aaa?gcc?ctc?cca?gcc?ccc?atc?gag?aaa?acc?atc?tcc?aaa?gcc 1203

Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala

385 390 395

aaa?ggg?cag?ccc?cga?gaa?cca?cag?gtg?tac?acc?ctg?ccc?cca?tcc?cgg 1251

Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg

400 405 410

gat?gag?ctg?acc?aag?aac?cag?gtc?agc?ctg?acc?tgc?ctg?gtc?aaa?ggc 1299

Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly

415 420 425

ttc?tat?ccc?agc?gac?atc?gcc?gtg?gag?tgg?gag?agc?aat?ggg?cag?ccg 1347

Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro

430 435 440 445

gag?aac?aac?tac?aag?acc?acg?cct?ccc?gtg?ctg?gac?tcc?gac?ggc?tcc 1395

Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser

450 455 460

ttc?ttc?ctc?tac?agc?aag?ctc?acc?gtg?gac?aag?agc?agg?tgg?cag?cag 1443

Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln

465 470 475

ggg?aac?gtc?ttc?tca?tgc?tcc?gtg?atg?cat?gag?gct?ctg?cac?aac?cac 1491

Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His

480 485 490

tac?acg?cag?aag?agc?ctc?tcc?ctg?tct?ccg?ggt?aaa?gcg?gat?cct?tcg 1539

Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Ala?Asp?Pro?Ser

495 500 505

aac?ctg?ctc?cca?tcc?tgg?gcc?att?acc?tta?atc?tca?gta?aat?gga?att 1587

Asn?Leu?Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile

510 515 520 525

ttt?gtg?ata?tgc?tgc?ctg?acc?tac?tgc?ttt?gcc?cca?aga?tgc?aga?gag 1635

Phe?Val?Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu

530 535 540

aga?agg?agg?aat?gag?aga?ttg?aga?agg?gaa?agt?gta?cgc?cct?gta 1680

Arg?Arg?Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

545 550 555

taaatcgata?ctcgag 1696

<210>3

<211>555

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: mouse-human fusion protein

<220>

＜221〉signal

<222>(1)..(23)

＜223〉L6 V κ signal peptide

<220>

＜221〉structural domain

<222>(24)..(133)

＜223〉G19-4 mouse anti human CD3 variable region of light chain

<220>

＜221〉polypeptide

<222>(134)..(148)

＜223〉(Gly4Scr) 3 joint polypeptide

<220>

＜221〉structural domain

<222>(149)..(270)

＜223〉G19-4 mouse anti human VH structural domain

<220>

<221>MISC_FEATURE

<222>(271)..(504)

＜223〉human IgG1 Fc structural domain (hinge area, CH2, CH3)

<220>

＜221〉stride film

<222>(505)..(555)

＜223〉people CD80 membrane spaning domain and tenuigenin tail

<400>3

Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser

1 5 10 15

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

20 25 30

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

35 40 45

Ser?Gln?Asp?Ile?Arg?Asn?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Asp

50 55 60

Gly?Thr?Val?Lys?Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Arg?Leu?His?Ser?Gly

65 70 75 80

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

85 90 95

Thr?Ile?Ala?Asn?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Phe?Cys?Gln

100 105 110

Gln?Gly?Asn?Thr?Leu?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Val

115 120 125

Thr?Lys?Arg?Glu?Leu?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly

130 135 140

Gly?Gly?Gly?Ser?Ile?Asp?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu

145 150 155 160

Leu?Val?Lys?Pro?Gly?Ala?Ser?Met?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Ser

165 170 175

Phe?Thr?Gly?Tyr?Ile?Val?Asn?Trp?Leu?Lys?Gln?Ser?His?Gly?Lys?Asn

180 185 190

Leu?Glu?Trp?Ile?Gly?Leu?Ile?Asn?Pro?Tyr?Lys?Gly?Leu?Thr?Thr?Tyr

195 200 205

Asn?Gln?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser

210 215 220

Ser?Thr?Ala?Tyr?Met?Glu?Leu?Leu?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala

225 230 235 240

Val?Tyr?Tyr?Cys?Ala?Arg?Ser?Gly?Tyr?Tyr?Gly?Asp?Ser?Asp?Trp?Tyr

245 250 255

Phe?Asp?Val?Trp?Gly?Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Asp?Leu

260 265 270

Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Ser?Pro?Pro?Ser?Pro?Ala

275 280 285

Pro?Glu?Leu?Leu?Gly?Gly?Ser?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro

290 295 300

Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val

305 310 315 320

Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val

325 330 335

Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln

340 345 350

Tyr?Asn?Ser?Tnr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

355 360 365

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala

370 375 380

Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro

385 390 395 400

Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr

405 410 415

Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser

420 425 430

Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

435 440 445

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr

450 455 460

Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe

465 470 475 480

Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys

485 490 495

Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Ala?Asp?Pro?Ser?Asn?Leu?Leu?Pro

500 505 510

Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile?Phe?Val?Ile?Cys

515 520 525

Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu?Arg?Arg?Arg?Asn

530 535 540

Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

545 550 555

<210>4

<211>556

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: mouse-human fusion protein

<220>

＜221〉signal

<222>(1)..(20)

＜223〉5B9 mouse anti human 4-1BB V κ signal peptide

<220>

＜221〉structural domain

<222>(21)..(133)

＜223〉5B9 mouse anti human 4-1BB VL structural domain

<220>

＜221〉polypeptide

<222>(134)..(149)

＜223〉(Gly4Ser) 3 joint polypeptide

<220>

＜221〉structural domain

<222>(150)..(271)

＜223〉5B9 mouse anti human 4-1BB VH structural domain

<220>

<221>MISC?FEATURE

<222>(272)..(505)

＜223〉human IgG1 Fc structural domain (hinge area .CH2, CH3)

<220>

＜221〉stride film

<222>(506)..(555)

＜223〉people CD80 strides film and tenuigenin structural domain

<400>4

Met?Arg?Phe?Ser?Ala?Gln?Leu?Leu?Gly?Leu?Leu?Val?Leu?Trp?Ile?Pro

1 5 10 15

Gly?Ser?Thr?Ala?Asp?Ile?Val?Met?Thr?Gln?Ala?Ala?Phe?Ser?Asn?Pro

20 25 30

Val?Thr?Leu?Gly?Thr?Ser?Ala?Ser?Ile?Ser?Cys?Arg?Ser?Ser?Lys?Ser

35 40 45

Leu?Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr?Leu?Gln?Lys

50 55 60

Pro?Gly?Gln?Ser?Pro?Gln?Leu?Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala

65 70 75 80

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

85 90 95

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

100 105 110

Cys?Ala?Gln?Asn?Leu?Glu?Leu?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys

115 120 125

Leu?Glu?Leu?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly

130 135 140

Gly?Gly?Gly?Ser?Ser?Gln?Val?Gln?Leu?Lys?Gln?Ser?Gly?Pro?Gly?Leu

145 150 155 160

Val?Gln?Ser?Ser?Gln?Ser?Leu?Ser?Ile?Thr?Cys?Thr?Val?Ser?Gly?Phe

165 170 175

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

180 185 190

Gly?Leu?Glu?Trp?Leu?Gly?Val?Ile?Trp?Ser?Gly?Gly?Ile?Thr?Asp?Tyr

195 200 205

Asn?Ala?Ala?Phe?Ile?Ser?Arg?Leu?Ser?Ile?Thr?Lys?Asp?Asp?Ser?Lys

210 215 220

Ser?Gln?Val?Phe?Phe?Lys?Met?Asn?Ser?Leu?Gln?Pro?Asn?Asp?Thr?Ala

225 230 235 240

Ile?Tyr?Tyr?Cys?Ala?Arg?Asn?Gly?Gly?Asp?Asn?Tyr?Pro?Tyr?Tyr?Tyr

245 250 255

Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser?Asp

260 265 270

Leu?Glu?Pro?Lys?Ser?Ser?Asp?Lys?Thr?His?Thr?Ser?Pro?Pro?Ser?Pro

275 280 285

Ala?Pro?Glu?Leu?Leu?Gly?Gly?Ser?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys

290 295 300

Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val

305 310 315 320

Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr

325 330 335

Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu

340 345 350

Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His

355 360 365

Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys

370 375 380

Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln

385 390 395 400

Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu

405 410 415

Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro

420 425 430

Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn

435 440 445

Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu

450 455 460

Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val

465 470 475 480

Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln

485 490 495

Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Ala?Asp?Pro?Ser?Asn?Leu?Leu

500 505 510

Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile?Phe?Val?Ile

515 520 525

Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu?Arg?Arg?Arg

530 535 540

Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

545 550 555

<210>5

<211>55

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence; Primer

<400>5

cgtcgatgag?ctctagaatt?cgcatgtgca?agtccgatga?gtcccccccc?ccccc 55

Claims (24)

1. produce the lymphocytic culture system of tumor response T, it comprises T cell from the cancer patients, antigen presenting cell, from body or allochthonous tumour cell with induce the immobilization antibody of polyclone activated T cell receptor.

2. the culture system of claim 1, wherein said antigen presenting cell is the monocyte from body, it produces described T cell and replys and break up.

3. the culture system of claim 1 is wherein saidly expressed at least a gene from tumour cell body or allochthonous is transfected, and described genes encoding stimulates the molecule of direct or indirect t cell response.

4. the culture system of claim 1, wherein said immobilization antibody combines with CD3 and CD28 acceptor.

5. composition, it comprises that coding can be at the polynucleotide of anti--CD3scFv of cell surface expression.

6. the composition of claim 5, wherein said polynucleotide encoding G19-4scFv.

7. composition, it comprises that coding can be at the polynucleotide of the anti-people 4-1BB scFv of cell surface expression.

8. the composition of claim 7, wherein said polynucleotide encoding 5B9 scFv.

9. composition, it comprise by coding can the gene institute transfection of the anti-CD3 scFv of cell surface expression from body or allogeneic tumour cell.

10. the composition of claim 9, wherein said from body or allogeneic tumour cell by G19-4 scFv transfection, described G19-4 scFv can be at cell surface expression.

11. a composition, it comprise by coding can the gene institute transfection of the anti-4-1BBscFv of cell surface expression from body or allogeneic tumour cell.

12. the composition of claim 11, wherein said from body or allogeneic tumour cell by 5B9 scFv transfection, described 5B9 scFv can be at cell surface expression.

13. treatment cancer patients's method, it comprises to the patient uses tumour cell, and described tumour cell is transfected can-CD3 scFv anti-at cell surface expression.

14. the method for claim 13, wherein said tumor cells expression G19-4.

15. treatment cancer patients's method, it comprises to the patient uses tumour cell, and described tumour cell is transfected can be at cell surface expression the anti-4-1 bb scFv.

16. the method for claim 15, wherein said tumor cells expression 5B9.

17. treatment cancer patients's method, it comprises to the patient uses plasmid, described plasmid comprise can-CD3 scFv anti-at cell surface expression sequence.

18. the method for claim 17, wherein said plasmid-encoded G19-4 scFv.

19. treatment cancer patients's method, it comprises to the patient uses plasmid, and described plasmid comprises can be in the sequence of the anti-4-1BB of cell surface expression.

20. the method for claim 19, wherein said plasmid-encoded 5B7 scFv.

21. isolating polynucleotide, it comprises the sequence that coding can-CD3 scFv anti-at cell surface expression.

22. isolating polynucleotide, it comprises that coding can be in the sequence of cell surface expression G19-4scFv.

23. isolating polynucleotide, it comprises that coding can be in the sequence of cell surface expression the anti-4-1 bb.

24. isolating polynucleotide, it comprises that coding can be in the sequence of cell surface expression 5B9scFv.

Images