CN1270630A - Method of treating leukocytes, leukocyte compositions and methods of use thereof - Google Patents

Abstract

The invention provides methods and compositions for treating leukocytes to arrest proliferation of the leukocytes and render them ineffective in eliciting graft-versus-host disease (GVHD), but effective to enhance engraftment of allongeneic donor cells and promote destruction of diseased cells or pathogens. Leukocyte compositions and methods of use of these compositions in alleviating disease, facilitating various types of immune reconstitution and immunotherapy, and enhancing engraftment of allogeneic donor cells, are also provided.

Description

Handle leukocytic method, leukocyte compositions and application method thereof

The reference of relevant application

The application requires the right of priority of the U.S. Provisional Patent Application series number 60/053,599 of application on July 21st, 1997.

Invention field

The present invention relates to breed but the cell composition of reservation function, and these preparation of compositions methods and applications.More particularly, the present invention relates to prepare the propagation that is used to transfer and suppress leukocytic method.

Background of invention

Implement the multiple pernicious and non-malignant hematologic disease of bone marrow transplantation (BMT) treatment, comprise leukemia, multiple myeloma, lymphoma, anaemia and immunodeficient disease, as severe severe combined immunodeficiency (SCTD), WAS and aplastic anemia.

Leukemia is the malignant tumour of hemopoietic tissue.These tumours can be divided into two kinds of main types: chronic and acute.Acute leukemia (for example is characterised in that undifferentiated cell colony, acute lymphoblastic leukemia (or " ALL ") and acute myelogenous leukemia (or " AML ")), and chronic leukemia shows more sophisticated morphology (for example, chronic granulocytic leukemia (or " CML ") and lymphocytic leukemia (or " CLL ")) usually.There is the new leukemia case of about 25700 examples nineteen ninety-five in the U.S., and wherein about 4500 examples are CML.The U.S. diagnosed out the new CML case of about 7000 examples in 1996, and wherein about 30% finally accepts allosome BMT (leukemia association: leukemia association website, 1997).Estimate that about 50% BMT patient suffers leukemic recurrence.

The general objects of leukemia treating is to suppress the propagation of abnormal morphology, and recovers " normally " hemopoietic in the marrow.Treatment plan can comprise the combination of chemotherapy, radiotherapy and bone marrow transplantation.In " genus " allogeneic bone marrow transplantation (allosome BMT) scheme, at first give the chemoluminescence treatment of leukaemic's severe bone marrow depression dosage.This treatment stage is used for eradicating all leukemia cells and my late grandfather thereof.After suppressing pathologic hematopoiesis physiology, adiaphorous marrow on the function is replaced with the anosis stem cell of allogeneic.Under top condition, the marrow of transplanting is beneficial to the recovery of normal polyclone hematopoiesis propagation.The improvement of disease control also depends on the immune-mediated reaction of graft to the leukemia cell, is called graft leukemia (GVL) effect.

Marrow is not unique source of hemopoietic progenitor cell.Peripheral blood and bleeding of the umbilicus also can be used as the source of stem cell.Referring to McCullough, blood ingredient of new generation, blood transfusion (Transfusion) 35:374 (1995).But systemic application nutritional factor (as granulocyte-G CFS) improves the propagation of peripheral hematopoietic stem cells, thereby produces the substitute of marrow, is used to gather in the crops hemopoietic progenitor cell.

Unfortunately, the major obstacle of successful BMT is the recurrence of graft versus host disease (GVHD), infection and primary disease.GVHD and infection cause M ﹠ M (" basis and clinical immunology " (the Basic and ClinicalImmunology) that transplants 10-30% in back preceding 100 days, the 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow (volume), Appleton ﹠amp; Lange, Norwalk CT, 1994).If the BMT failure, the unrestricted propagation of tumors remaining cell can cause " recurrence ".Referring to people such as Slavin, " with the immunotherapy of immunocompetent lymphocytes and the minimum remaining disease of active cells factor pair thereof ", cancer research (Cancer Investigation), 10:221 (1992).Past therapeutic treatment is in this case selected very limited.For example, the widespread use of bone marrow transplantation and/or cytotoxic drug is relevant with extremely low survival rate for the second time.

Yet, by drawing a kind of therapeutic strategy to transplanting biological understanding more fully.To there being or not having studies show that of BMT under the T cells exclude, for the leukemia of the treatment that can be used for the minimum remaining disease of CML patient or " comprehensively " recurrence was treated, the T cell was important.Based on this understanding, become a kind of generally acknowledged treatment with donor white corpuscle input (DLI) treatment to Leukemia Patients after the bone marrow transplantation.Referring to people such as Kolb, blood (Blood) 76:2462 (1990); Lim, people such as S H., U.S.'s hematology magazine (Am.J.Hem.) 54:61-67,1997; Giralt, people such as S.A., modern viewpoint (Cur.Op.Onc.) 8:89-95 of oncology, 1996.The leukocytic adoptive immunotherapy that also has been used as after bone marrow transplantation is failed that transfers is induced permanent alleviation.Referring to, J.Mccullough, blood ingredient of new generation, blood transfusion, 35:374 (1995).DLI is effective especially for the treatment of CML, but also can be used for the treatment of AML, ALL and CLL.DLI also has been used for the treatment of myelodysplasia (MDS), non_hodgkin lymphoma, Hokdkin disease and multiple myeloma.As if the cell of input cause the immunne response to the acceptor malignant cell.According to the pcr analysis of bcr-abl being reset (Philadelphia chromosome), about 70% recurrence is that the CML patient of chronic phase reaches persistent cytogenetics release behind DLI, and becomes (Drobyski, the people such as W.R. of remaining disease-negative, blood 82:2310-2318,1993).

Yet this method has danger, because GVHD takes place 80% among the patient, 56% osteomyelodysplasia takes place.This is two major causes of treatment failure, causes reaching 20% reaction patient's death (Champlin, people such as R., hematology journal (Act.Haemat.) 95:157-163,1996).GVHD causes by the donor T lymphocyte that exists among BMT that transfers or the DLI, breeds and is settled in host tissue.In case propagation, donor T cell is promptly attacked host tissue, causes pathological symptom.

The elimination of disease is considered to owing to the immune response of graft to tumour cell behind the DLI, i.e. GVL effect (Barrett, people such as J., the modern viewpoint of oncology, 8:89-95,1996).The mechanism of GVL effect is not got clear as yet fully.It is the T cell realization that origin comes from donor, because the BMT of T cells exclude causes the more high incidence of leukemia relapse.Immunne response is that this effect of generation is necessary, and BMT compares with allogeneic, and higher recurrence rate has proved this point (Duell, T., international medical science annual report (Ann.Int.Med.) 126:184-192,1997) behind the homology BMT in experiment.In Japan, crowd's high homogeneity causes invalid DLI treatment, because donor and host are not fully allochthonous (Takahashi, people such as K., lancet (Lancet) 343:700-702,1994) at random.

Clinical effectiveness shows that the GVL effect can be distinguished with pathogenic GVHD.Patient with the allogeneic BMT of T cells exclude treatment shows higher recurrence rate than the patient with BMT and standard GVHD prophylactic treatment, even when control GVHD sickness rate is counted still (Horowitz, people such as M.M., blood 75:555-562,1990) like this.The case that many reports are also arranged is observed the release of CML and is not had the morbidity (Duell, T., international medical science annual report 126:184-192,1997) of GVHD.In animal model, the differentiation of GVHD and GVL be attainable (referring to, for example, Johnson, people such as B.D., bone marrow transplantation (Bone Marrow Transplant.) 11:329-336,1993; Glass, people such as B., Britain's hematology magazine (Br.J.Hem.) 93:412-420,1996; Johnson, people such as B.D., blood 85:3302-3312,1995).

For fear of Acute GVHD, remove donor T lymphocyte or the mature lymphocyte that in BMT, exists with several technology.Yet current do not have effective marrow to handle to avoid GVHD fully, because in most of the cases the GVL effect is weakened, cause higher recurrence rate (Kantarjian, people such as H.M., blood 87:3069-3081,1996).Some experimental technique of people and animal model comprises, the eliminating of T cell subsets, UVB shines (Greenfeld, people such as J.I., surgery research magazine (J.Sur.Res.) 60:137-141,1996), or the T cells exclude adds a certain amount of T cell, the shortage (Drobyski of the GVHD of a certain amount of T cell of titration subsequently again, W.R. wait the people, blood, 82:2310-2318,1993).In a kind of strategy, with a kind of " suicide gene " transfection donor T cell, this gene provides the herpesvirus thymine deoxyriboside kinase of the susceptibility of medicine gancyclovir (HSV-tk) gene.If observe the GVHD symptom, then give patient's gancyclovir to eliminate the T cell in vivo, up to resolution of symptoms (Bordignon, people such as C., human gene therapy (Hum.Gen.Ther.) 6:813-819,1995; People such as Bonini C., science (Science) 276:1719-1724,1997).Another kind of " suicide gene " method is used the FAS gene of coding apoptotic signal transferrin (Ariad Pharmaceuticals).In the FAS system, induce FAS to produce with a kind of dimerization agent such as FK1012, kill and wound the donorcells of expressing this gene.

" suicide gene " also has many significant disadvantages although perhaps method is attractive from treating the viewpoint of eliminating unwanted T cell after the remaining cancer.At first, use the GVHD symptom signal of pharmacological agent to start with.Therefore, GVHD should be able to begin and must be able to close.Secondly, the medicine that is used for eliminating the T cell must systemic administration, thereby whole health being contacted with medicine reach makes the controlled necessary certain hour of GVHD.Gancyclovir has toxic side effects, and HSV-tk albumen is immunogenic.In addition, because the patient that needs protection avoids the severe form of GVHD, so the immunosuppression prevention is still indispensable.Equally, in some chronic GVHD example, prove that using of gancyclovir is not (people such as Bonini C., science 276:1719-1724,1997) in full force and effect.This perhaps be since this method prevent the explanation misread, promptly having only proliferating cells is the gancyclovir sensitivity.In addition, even the proliferating cells auxiliary effect (Good Samaritan effect) of escaping gancyclovir of enzyme that also can provide by near the normal cell that exists the contiguous HSV-tk cell.

For the FAS system, shortcoming comprises synthetic difficulty and induces the insoluble of dimerization agent that FAS produces.The proteic combination of FKBP can be limited the available medicine among dimerization agent and the host.

In addition, technology relevant with gene therapy and practice are considered to make and are difficult to carry out application with control techniques.The generation of transfectional cell is perplexed by poor efficiency, and this makes when the GVHD sign occurring, and the ability of the homogeneity of processed cell colony and total kill cell becomes problem.Cell must can be bred time several weeks, and this means between harvested cell and transfectional cell can be used for time of infusion has a delay.At last, and also be most important ground, the cell in the input human body may contain the genetic material of modification, and it might have deleterious effect for a long time to the host.

Ionizing rays (γ-radiation or X-radiation) or UV line (UVC λ=200-280nm; UVB λ=280-320nm; UVA λ=320-400nm) has been used to the processing blood goods and has come dna damage in the inducing cell.The method of the GVHD prevention that current generally acknowledged being used to transfused blood relevant is γ-radiotreatment (2500 cGy).γ-radiating the clinical dosage that is used to prevent TA-GVHD causes T cell alive to reduce 10 ⁵-10 ⁶Doubly.Yet γ-radiation and ionizing rays are not special for nucleic acid usually.Because its high-energy, γ-radiation and UVC are also attacked protein and other cellular constituent, cause tangible non-specific damage (Deeg, people such as H.J., hemocyte (Blood Cells) 18:151-162,1992).In order to prevent GVHD, the viability that keeps BM progenitor cell and original hemopoietic stem cell with the propagation of UVB elimination rat lymphocyte is used for implanting.At 4000J/cm ²The UVB treatment dosage time, CTL is active to be reduced but still has (Gowing, people such as H., blood 87:1635-1643,1996).Yet the UVB radiation also causes tangible cell injury, particularly (Gowing, people such as H., blood 87:1635-1643,1996 when using the lamp of wide emmission spectrum; Pamphilon, people such as A.A., blood 77:2072-2078,1991).Reported the surface molecular chemically modified and the film rupture that cause low cell survival and immune response inducing, this perhaps is because UVB induced protein and the lipid ability of the chemically changed of unsaturated link(age) especially.These restrictions are because UVB is not only selective to nucleic acid, but modify this fact of any chemical group that absorbs 280-320nm.

Be used for human leukocyte processing, the demonstration of the UVA radiation in the presence of photosensitizers 8-methoxypsoralen (8-MOP), the stimulation ability and the propagation (Kraemer that in external mixed lymphocyte culture reaction, suppress peripheral blood leucocyte, K.H. wait the people, dermatological studies magazine (J.Inv.Derm.) 77:235-239,1981; Kraemer, people such as K.H., dermatological studies magazine 76:80-87,1981).Yet, described in these reports, UVA adds psoralene and handles inhibition (Gruner, the people such as S. who causes surface antigen expression, cytokine synthetic (IL-1, IL-6, IL-8 and INF) and cytokines mRNA to be transcribed, tissue antigen (Tiss.Ant.) 27:147-154,1986; Neuner, people such as P., photochemistry and photobiology (Photochem.Photobiol.) 59:182-188,1994).Therefore, the photochemical treatment described in the former research (PCT) condition is although suppress propagation and reduce non-specific cell injury, the also immunologic function of deactivation T cell; These treatment condition can not produce the cell that any immunoprotection effectively is provided or induces the GVL effect in order to transfer purpose.At last, spleen and myelocytic UVA are added the reduction (Ullrich, S.E., dermatological studies magazine 96:303-308,1991) that the 8-MOP processing has been successfully used to GVHD in the mouse model.As former all reports, this research is at the leukocytic GVHD of donor aspect, and do not provide immunoprotection or GVL function for white corpuscle.

Obviously, have the needs to novel method, the GVHD that suppresses between allohistocompatibility's obstacle induces, and does not need to contact with the system of medicine.This method can provide certain immunoprotection to the individuality of non-responsiveness, and/or allows the killing and wounding of residual cancer, but should be able to prevent to import the propagation of cell in the host.

In this description and require the present invention of patent protection, by providing preparation not induce GVHD but keep the leukocytic method of leukocyte function, at and overcome these and other problem relevant with prior art.By the following detailed description, this and other advantage that method of the present invention had will be obvious.

Summary of the invention

The invention provides a kind of effective ways, be used for the white corpuscle of treatment of allogeneic donor, keep viability and effective white corpuscle that promotes disease cell or pathogenic agent destructive immunologic function thereby generation can not be bred.Because treated white corpuscle can not be bred, so they can not induce GVHD (during in homology or autotransfusion when introducing the allogeneic host, GVHD is not a problem), therefore, they can be used for the DLI for various clinical indication especially treatment for cancer ideally, and provide immunologic function for the Mammals of non-responsiveness.

Many aspects of the present invention comprise following aspect:

A kind of isolated cells colony, it contains a kind of leukocyte population, and wherein the part of this leukocyte population is not bred, and makes this cell colony can not cause graft versus host disease (GVHD) in the allogeneic host; And the part of this leukocyte population keeps immunologic competence, comprises disease cell or the pathogenic agent destructive ability of promoting.Preferably, at least 90% white corpuscle is not bred in this colony.

The feature of the leukocyte population of previous embodiments further is: 1) preferably comprise T cell, NK cell and antigen presenting cell; 2) after suitably stimulating, can synthesize and/or secrete cytokines, as IL-2, IFN-γ, IL-10 and GM-CSF; 3) can expression characteristic be the surface markers of t cell activation and immunologic function, such as especially CD4, CD8, CD16 and CD56; And 4) compare with untreated leukocyte population, show killing ability that improves and the ratio of breeding function.

Leukocyte population of the present invention can not be bred generally, but keeps immunologic competence.This leukocyte population especially will have following activity:

1. promote the destruction of disease cell, infected cell or pathogenic agent.

2. help the implantation of second kind of cell colony.Second cell colony can be a kind of thin

Born of the same parents' suspension or organized cell aggregation are as a block organization or organ.These cells can wrap

It is thin for example to draw together hematopoietic cell, myelocyte, white corpuscle, medullary cell, islet cells, liver

Born of the same parents, neuronal cell, myocardial cell, mesenchymal cell and endotheliocyte.

3. promotion immunologic reconstitution.

4. immunotherapy.

5. mix the treatment of chimerism (mixed chimerism).

6. promote graft leukemia (GVL) effect.

In one embodiment, this leukocyte population is effective for the destruction that promotes cancer cells.Preferably, cancer cells is selected from: chronic lymphocytic leukemia (CML) cell, chronic Myelomonocyte leukemia (CmML) cell, lymphocytic leukemia (CLL) cell, acute myelogenous leukemia (AML) cell, acute lymphoblastic leukemia (ALL) cell, multiple myeloma (MM) cell, He Jiejin lymphomas cell and Fei Hejie lymphomas cell.In preferred embodiments, cancer cells is chronic lymphocytic leukemia (CML) cell or multiple myeloma cells.

In another embodiment, this leukocyte population can promote the destruction of disease cell effectively, and wherein the disease cell is a kind of infected cell.Infected cell comprises by the cell of virus infection.Preferably, the virus of cells infected is selected from cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) and kaposi sarcoma-associate herpesvirus.

In the third embodiment, this leukocyte population is effective for the destruction that promotes pathogenic agent.Pathogenic agent comprises bacterium, fungi and parasite.

In another embodiment, this leukocyte population is effective for the implantation that promotes second kind of cell colony.The cell that is used to implant can be, for example, and hematopoietic cell, myelocyte, white corpuscle, medullary cell, islet cells, liver cell, neuronal cell, myocardial cell, mesenchymal cell and endotheliocyte.In addition, second of implantation kind of cell colony can comprise solid organ or its part.

The present invention also provides the subgroup with the above-mentioned leukocyte population of several different methods screening.This subgroup comprises lymphocyte and T lymphocyte; And can obtain these subgroups by the positive and the negative selection of expressing based on for example surface markers.Preferred surface markers comprises CD8, CD4, CD16 and CD56.The method of screening leukocyte population subgroup comprises white corpuscle electrophoresis (leukophresis) and red corpuscle removal from whole blood.

In another embodiment, the invention provides the multiple therapy methods of using above-mentioned cell colony, comprise donor leukocyte infusion, white corpuscle return add, the treatment of immunologic reconstitution, adoptive immunotherapy, mixing chimerism and strengthen the method that second kind of transplanted cells colony implants.For strengthening the method that second kind of transplanted cells colony implants, can be before second kind of cell colony be transplanted, transplant simultaneously or afterwards cell colony of the present invention is introduced among the host.

The present invention also provides the method for preparing treated leukocyte population, and wherein this leukocyte population is not bred generally, and can not cause graft versus host disease (GVHD) in the allogeneic host, and this method comprises the following steps:

I) provide a kind of sample that contains leukocyte population; With

Ii) make this sample and can combine with a certain amount of, make that this compound can be per 10 with the compound of nucleic acid form covalent linkage ⁸Form about 1-10 in the individual white corpuscle genomic dna base pair ⁴Individual adducts, thus propagation suppressed but the reservation immunologic competence, comprise that leukocyte population promotes disease cell or pathogenic agent destructive ability.

In a kind of preferred embodiment, this compound is to be enough to per 10 ⁸Form about 5-10 in the individual white corpuscle genomic dna base pair ³The amount of individual adducts exists.Preferably, this method causes in the treated leukocyte population at least 90% T cell proliferation to be suppressed.

Preferably, can further comprise with the compound that nucleic acid forms covalent linkage a kind of can with the non-covalent bonded nucleic acid binding moiety of nucleic acid.

In one embodiment, this compound comprises: nucleic acid binding moiety; Can form the part of covalent linkage with nucleic acid reaction; And a kind of fragility linker of covalently bound nucleic acid binding moiety and effector part.Preferably, nucleic acid binding moiety is a kind of aromatics insert and effector partly is a kind of mustard seed base (mustard).

In a kind of preferred embodiment of the method for the treated leukocyte population of preparation, but can contain a kind of photoactivation part with the compound that nucleic acid forms covalent linkage, it can form covalent linkage with nucleic acid behind electromagnetic stimulation.When but use has the compound of photoactivation part can light-activated compounds the time, this method further comprises the following steps: to make the white corpuscle sample with this compound to be exposed under the light, but, thereby but cause photoactivation part and white corpuscle genomic dna to form covalent linkage with photoactivation photoactivation part.

In a kind of embodiment of preceding method, be selected from: furocoumarin(e), actinomycin, anthracene nucleus ketone, anthramycin, benzodipyrane ketone, fluorenes, Fluorenone, single star fat indigo plant, norphillin A, organic dye but have photoactivation compound partly; Phenanthridines, thiodiphenylamine sulfosalt, azophenlyene, thiodiphenylamine, triazobenzene, quinoline and thioxanthone, acridine and ellipticine.Preferred furocoumarin(e) is a psoralene.Preferred psoralene comprises PAP, 8-methoxypsoralen (8-MOP), 4 '-aminomethyl 4,5 ', 8-trimethylpsoralen (AMT), 5-MOP and trioxa quinoline (trioxalene) 4,5 ', 8-trimethylpsoralen.

When being used for handling leukocytic compound and being psoralene, this psoralene is preferably with 10 ^-4The concentration of-150 μ M exists, and to make the white corpuscle sample be exposed to wavelength be 200-450nm, preferably under the ultraviolet ray of 320-400nm.Preferably, with 10 ^-3-100J/cm ²Dosage provide the ultraviolet ray.The white corpuscle sample will be exposed to following 1 second to 60 minutes for some time of ultraviolet ray.

In a kind of preferred embodiment, prepare the method for treated leukocyte population according to above embodiment, use general formula:

Psoralene (being called as S-59 herein) and salt thereof.S-59 will be with 10 ^-4-150 μ M, more preferably 10 ^-3The concentration of-150 μ M is used.Make that to be exposed to wavelength with S-59 blended white corpuscle sample be 200-450nm, preferably under the ultraviolet ray of 320-400nm.Preferably will be exposed to dosage with S-59 blended white corpuscle sample is 10 ^-3-100J/cm ², 3J/cm more preferably ²Ultraviolet ray under.For photoactivation S-59, preferably the white corpuscle sample is exposed to following 1 second to 60 minutes of ultraviolet ray, for some time of 1 minute preferably.Preferably with every milliliter of 10-10 ⁹Individual cell, more preferably every milliliter 10 ²-10 ⁸Individual cell, most preferably every milliliter 2 * 10 ⁶The cell density of individual cell provides the white corpuscle sample.

The leukocyte population that produces according to preceding method is provided, comprises the leukocyte population of handling with S-59 especially.

Of the present invention more on the other hand for promoting disease cell or pathogenic agent destructive method, comprise that the leukocyte population that will produce with aforesaid method mixes mutually with the homogeneous variant cell colony of containing disease cell or pathogenic agent.This method can be carried out in external or body.In a kind of preferred embodiment, leukocyte population is mixed in vivo with the homogeneous variant cell colony of mammalian hosts by the input of donor white corpuscle in the host.Preferably, the mammalian hosts of suffering from leukemia behind the BMT or multiple myeloma recurrence is implemented donor leukocyte infusion.

In a kind of preferred embodiment that promotes disease cell or pathogenic agent destructive method, the disease cell is a cancer cells.Comprise the cancer cells that derives from following cancer: chronic lymphocytic leukemia (CML) cell, chronic myelomonocytic leukemia (CmML) cell, lymphocytic leukemia (CLL) cell, acute myelogenous leukemia (AML) cell, acute lymphoblastic leukemia (AML) cell, multiple myeloma (MM) cell, He Jiejin lymphomas cell and non_hodgkin lymphoma cell.In a kind of the most preferred embodiment, cancer cells is chronic myeloid leukemia cells or multiple myeloma cells.

Another kind of preferred cancer cells is the cancer cells that is selected from breast cancer cell, lung carcinoma cell, ovarian cancer cell, testicular cancer cell, prostate cancer cell, colon cancer cell, melanoma cells, kidney cancer cell, neuroblast oncocyte, a cancer cells and neck cancer cells.

In the another kind of preferred embodiment that promotes disease cell or pathogenic agent destructive method, the disease cell is a kind of infected cell.Preferred infected cell comprises by the cell of a kind of virus such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) or kaposi sarcoma-associate herpesvirus infection.

In the another embodiment again that promotes disease cell or pathogenic agent destructive aforesaid method, using stimulates leukocyte population to disease cell or one or more special epitopes of pathogenic agent, enlarges the quantity to the cytotoxic T cell of antigen-specific.Stimulate or the T cell amplification of antigen-specific can be in vivo, exsomatize or carry out external.The method of carrying out the body internal stimulus is separate leukocyte population from donor before, uses disease cell or the special antigen inoculation white corpuscle donor of pathogenic agent.In disease is in the situation of CML, preferably uses the bcr-abl antigenic stimulation leukocyte population of CML cell.In the situation of multiple myeloma, can inoculate the white corpuscle donor with patient myeloma cell's idiotype antigen.

In the another embodiment again that promotes disease cell or pathogenic agent destructive preceding method, stimulate leukocyte population with a kind of mitogen.Preferably use a kind of mitogenesis composition such as phorbol myristate acetate (PMA) adds ionomycin or phytohemagglutinin stimulates external.

The accompanying drawing summary

Fig. 1 shows with S-59 (square), the AMT (trilateral) of different concns and 8-MOP (circle) photochemical treatment (PCT) back (UVA=1J/cm ²) minimizing (referring to embodiment 1) of proliferative T cell.

Fig. 2 is presented in the MLR test effector cell's that the S-59 with different pharmaceutical dosage handles ³The H-thymidine mixes (referring to embodiment 2).

Fig. 3 shows the IL-2 generation level (referring to embodiment 2) of effector cell in MLR with the S-59 photochemical treatment of different pharmaceutical dosage.

Fig. 4 shows the IFN-γ generation level (referring to embodiment 2) of effector cell in MLR with the S-59 photochemical treatment of different pharmaceutical dosage.

Fig. 5 shows S-59 and the 0.5J/cm with different concns ²UVA to the white corpuscle PCT among the PC after, with the IL-8 level (referring to embodiment 2) of the generation that the time becomes behind the PCT.

Fig. 6 shows AMT and the 1J/cm with various dose ²Behind the UVA PCT, with the level (referring to embodiment 3) of the CD69 marker expression that the time changes after the processing.

Fig. 7 is presented among the PC formation with psoralene-dna adduct after the multiple psoralene photochemical treatment.S-59 (square); AMT (trilateral); 8-MOP (circle); 1.9J/cm ²UVA (referring to embodiment 3).

Fig. 8 shows that S-59+UVA handles the influence to processed cell proliferation, basis in the MLR test ³The H thymidine mixes mensuration.NR represents untreated cell; The cell that shines with UVA when UV represents not have S-59.Also shown in the presence of the S-59 of three kinds of different concns and used 3J/cm ²The result who obtains during the UVA irradiating cell.See embodiment 14 for details.

Fig. 9 shows to make with anti-CD 3 antibodies and measures behind the processed cell-stimulating that S-59+UVA handles the influence to processed cell proliferation.See embodiment 14 for details.

Figure 10 A shows with the S-59+UVA photochemical treatment and at the human PBMC's of MLR moderate stimulation IL-2 and produces.Measure by sandwich ELISA.See embodiment 14 for details.

Figure 10 B shows with the S-59+UVA photochemical treatment and at the human PBMC's of MLR moderate stimulation IFN-γ and produces.Measure by sandwich ELISA.

Figure 11 A show with anti--CD3 activate the back in different time (hour) CD69 in the treated and control cells measured expresses.

Figure 11 B show with anti--CD3 activate the back in different time (hour) CD25 in the treated and control cells measured expresses.

Figure 12 show with anti--CD3 activate the back in different time (hour) CD40L in the treated and contrast untreated cell measured expresses.

The chemical treatment of Figure 13 display light, the leukocytic cytotoxic t cell activity of activated, according to the treated white corpuscle target cell of incubation altogether ⁵¹Cr discharges and measures.

Figure 14 shows the splenocyte accept MHC-mispairing bone marrow transplantation and S-59+UVA and to handle, then after transplanting three days in the exposure mouse with leukemia cell's attack, the mensuration of mean body weight.As shown in the figure, in the presence of 0.01 μ M S-59, make splenocyte be exposed to different time length under the UVA.

Figure 15 is presented at or accepts when not having treated white corpuscle infusion the active analysis of GVL in the mouse that the leukemia of bone marrow transplantation attacks.GVL is active to prove (having provided survival ratio in the bracket) with no leukemia percentage survival.

Figure 16 shows the analysis of effector cell's multiplication capacity that S-303 handles, and cultivates the back altogether 6-7 days with the allogeneic irritation cell of γ-deactivation in MLR, according to ³The H-thymidine mixes mensuration.

Figure 17 shows the IFN-γ level in the MLR supernatant liquor, wherein uses the S-303 Treatment Effects cell of different concns before cultivating altogether.

The detailed Description Of The Invention abbreviation

Use following abbreviation:

BMT: bone-marrow transplantation

DLI: donor leukocyte infusion

LDA: limiting dilution analysis

MLR: mixed lymphocyte reaction (MLP)

PA: photochemistry suppresses; PCT: photochemical treatment; PI-DLI: the donor leukocyte infusion of photoinactivation; PUVA: psoralen and ultraviolet light,long wave irradiation.

PAP:4 '-and the psoralen that replaces of 5 '-primary amino radical

S-59: the psoralen that a kind of primary amino radical with following formula replaces:

PBSC: peripheral hematopoietic stem cells

PC: platelet concentrate

TA-GVHD: transfer relevant graft versus host disease

PHA: phytolectin definition

" allogeneic " refers to the relation that exists between the members different in the heredity mutually of the same race, and namely these members are not homologies.

" host " can with " acceptor " exchange interaction, refer to the leukocytic mammalian receptors of allogeneic donor. Usually, the host is the people, but also comprises other mammal, such as mouse, dog, cat, monkey, horse etc.

" leucocyte " refers to any leucocyte, comprises the pedigree CFU-GM. " antigen presenting cell " comprises macrophage, and the dendritic cells of a small amount of existence in peripheral blood, and their precursory cells separately. Leucocyte be present in the blood circulation and marrow in, and the myelocyte formation of reticuloendothelial system, lymph and netted position.

" leukocyte population " refers to contain the leukocyte population that surpasses a kind of leucocyte cellular type as used herein, and comprises at least T cell, antigen presenting cell and NK cell.

" donor leucocyte " refers to for host animal is not endogenous and derive from a kind of leucocyte of allogeneic donor. Use for external and non-human, leukocyte population and donor leucocyte can derive from mammal, comprise rodent, rabbit, dog etc.

" purifying " meaning is, leukocyte population is separated from health, and processed so that be substantially free of non-leukocytic cellular type, and does not preferably contain other pollutant, such as the cell relic that exists in the leucocyte source, this source is generally peripheral blood, marrow even splenocyte. Preferably, the purifying leukocyte population contains it and is less than 13% red blood cell (RBC), more preferably is less than 5%, even more preferably is less than 1%. In a kind of the most preferred embodiment, hematocrit levels is lower than 0.5%. The leucocyte purification process has below been described.

" treated leucocyte " refers to be exposed to or contacts a kind of leucocyte that can form with nucleic acid the compound of one or more covalent bonds. Leucocyte can be by PCT or with alkylating agent compound " processing ". " photoinactivation " or " photochemistry suppresses " (PA) or " PCT suppresses " leucocyte PCT leucocyte of referring to even still can not breeding and do not mean that leucocyte is being inactivated aspect protein expression and the immunologic function after stimulation.

Leukocyte population of the present invention contains but is not limited to " not breeding " cell. If it is suppressed to copy middle cell at DNA, thereby even after stimulating with reagent such as mitogen, cell factor, antigen, antibody or other propagation stimulus, still can not divide and produce new cell, then this cell is " not breeding ". Method of the present invention causes in the preparation every kind of leukocytic copying all to suppress optional. For the present invention, as long as the small part leucocyte that can breed is not enough to cause GVHD in the allogeneic host, then in the colony of purifying at least 90%, more preferably at least 95% the leucocyte, leukocyte population breed be suppressed just enough. In a kind of the most preferred embodiment, in the colony 99% or more leucocyte do not breed. For example, by limiting dilution test (LDA) or pass through separately³The H-thymidine mixes method, or as described in the following Examples as the part of MLR test, can measure proliferation activity. In a kind of the most preferred embodiment, the T cell in the colony is suppressed to the lower limit that LDA detects by propagation. Shown the ability that the treated leucocyte of these result of the test indications is bred in vivo.

" graft versus host disease " or " GVHD " result from the clone expansion that is present in the donor T cell that transfers among BMT or the DLI, breed and be settled in host tissue. The generation and the order of severity and the existence relevant (referring to Kernan, the people such as N.A., blood 68:770-773,1986) that can clone the T cell of GVHD have been confirmed. With the patient of donor HLA-coupling in, GVHD results from minor histocompatibility antigen. The morbidity of GVHD is the shortage owing to immunologic function among the immunosuppressant patient, and this is the necessary condition of BMT success. Donor T cell can not bred in this environment under fire, and attacks host tissue, causes pathological symptom. On cellular level, host's antigen presenting cell and donor T cell interact in the scope of major histocompatibility complex (MHC) I and II, and induce it to the activation of the cell that carries host specificity (less important) antigen. This causes the clonal expansion of activated T cell, and it attacks host tissue and the release cells factor. The cell factor of T emiocytosis also activates multiple other effector cell of host, and this other generation by cell factor (cell factor tide) increases the weight of tissue damage. Referring to, Burakoff for example, the people such as S.J., graft versus host disease immunology, Pathological Physiology and treatment. In Brinkhous KMS, S.A. (volume): " hematology " be the 12nd volume (front page) (Hematology), New York, Marcell Dekker, Inc., 1990,725 pages.

When diagnosis had GVHD, the immunosuppressive drug that usually gives patient's high dose suppressed GVHD. Yet these medicines also make the leucocyte of transplanting invalid aspect GVL, and the patient can be recurred and is leukaemia.

Leukocyte population causes that the ability of GVHD can be as measuring as described in following examples 4 in vivo, perhaps by limiting dilution test (LDA) at external test, perhaps as described in following examples 5, measure by MLR. Measure according to LDA, if the cell quantity of the cloned T in the colony is 10 of existence in the leucocyte control population (positive control of GVHD and propagation) that is untreated^-3-10 ^-4, judge that then this leukocyte population can not cause GVHD. In vivo, the shortage of GVHD clinical symptoms (embodiment 4 described symptoms) shows that this colony can not cause GVHD in the allogeneic acceptor of leukocyte population.

If a kind of leukocyte population comprises such leucocyte, it can directly or indirectly participate in effectively killing and wounding or to remove the immune response of target disease cell (or pathogen) or restriction disease cell (or pathogen) propagation from health, then think this colony " be effective for the destruction that promotes disease cell or pathogen ". Be not that the every kind of leucocyte that exists in the leukocyte population can both promote to destroy, but this colony should be effective in this regard generally.

Will be appreciated that disease cell or cause of disease physical efficiency are destroyed by any mechanism. Can mediate the leukocytic method of specific mechanism restriction processing of destroying by treated leucocyte is not purpose of the present invention. The disease cell can be destroyed by cytolysis by T cell or NK cell, and can be killed and wounded by other mechanism, as is induced to experience apoptosis. Disease cell or pathogen also can be relied on the cell-mediated cytotoxicity (ADCC) of antibody, the phagocytosis of macrophage destroys, or removed by reticuloendothelial system by naturally occurring arbitrary immunologic mechanism in the mammal. The host cell that relies on by killing and wounding its survival can destroy the pathogen that resides in the host cell indirectly. Learn textbook such as " Preclinic and clinic immunology " the 8th edition at standard immunoassay, Daniel P. Stites, Abba I.Terr and Tristram G.Parslow (volume), Appelton ﹠ Lange, Norwalk CT, in 1994, immune system has been discussed has been made body break away from the mechanism of malignant cell, infected cell or pathogen. The donor leucocyte that " effectively " processes can be a kind of effector cell, such as cytotoxic T cell (CTL), NK cell or a kind of macrophage, and its direct killing target disease cell or pathogen, or induce disease cell experience apoptosis. In addition, by stimulating other leucocyte, for example come from the marrow of transplanting, the leucocyte of former DLI, or host's self leucocyte, treated donor leucocyte can mediate destruction, thereby kills and wounds or remove. Can stimulate other leucocyte by surface antigen expression, cytokine secretion or any suitable mechanism. Cytolysis can occur in the mechanism through MHC-restriction and/or non-MHC restriction (such as the NK cell).

The usefulness that leukocyte population promotes disease cell or pathogen to destroy can be by multiple test determination, MLR as described in the following Examples or⁵¹The Cr release test. For example, according to⁵¹The ability that the mediation leukaemia kills and wounds in the Cr release test has proved cytolytic usefulness. For the present invention, if in suitable test (for example⁵¹The Cr release test), leukocyte population shows the molten cell ability than high at least about 20% the level of negative control colony, thinks that then this colony is effective to the destruction that promotes the disease cell. Below provide for disease cell and the pathogen type of killing and wounding.

Usually, promote the usefulness of destroying to need leukocyte population to keep certain synthetic level of protein, produce and secretion so that comprise the albumen mass-energy of signal transducers (such as cell factor). Preferably, on plasma membrane, also there is surface antigen to comprise acceptor, the expression of adhesion molecule and costimulatory molecules. Cell survival and film integrality are important for mediation GVL effect. " cell survival " is defined herein as in the circulation is survival rate in blood flow and other tissue.

Although the generation of some cell factor can be suppressed, preferably the cell factor generation remains on at least 70%, more preferably 80% before the above-mentioned compound treatment even more preferably is higher than 90%, most preferably is higher than 95% level. Preferably, leukocyte population is included in the leucocyte that stimulates lower at least secreting leukocytes mesonium-2 (IL-2) and interferon-γ (IFN-γ). Because the clearly effect of these cell factors in t cell activation and GVHD/GVL, they are correlated with. Other relevant cell factor comprises granulocyte-macrophage colony stimutaing factor (GM-CSF) and interleukin 10 (IL-10). For example, can easily measure the secretion of cell factor with the described cytokine assay kit of the following part of present embodiment (for example, the kit of R ﹠ D Systems).

The surface antigen that leucocyte in the purifying leukocyte population is expressed (being also referred to as surface markers) depends on specific cellular type. Preferably, this colony comprises the leucocyte of expressing following surface antigen: CD2, CD28, CTLA4, CD40L (gp39), CD18, CD25, CD69 (lymphocyte activator mark) and CD16/CD56, antigen, known they participation T cells interaction and immunologic function relevant with the NK cell-stimulating. Preferably, leucocyte is also expressed other surface markers, comprises MHC I class and II class, CD8, CD4, CD3/TcR (φt cell receptor), adhesion molecule such as CD54 (ICAM-1), LFA-1 and VLA-4, and other co stimulatory molecule. Available many measure method can detect and measure the expression of surface antigen, for example analyzes to use by standard FACS-SCAN the special antibody staining cell of specific antigen is also detected by the antibody of direct or indirect mark. Other method known in the art comprises immunoprecipitation and the Western blotting of surface antigen.

" disease cell " refers to the cell of cancer cell, infected cell or any type pathology as used herein, and it is interior or external that they may be present in body. Cancer cell or malignant cell may derive from any type cancer, be any tissue or cellular type source. These pernicious or cancer cells include but not limited to the cell of following malignant tumour: leukaemia comprises chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL); Huppert's disease (MM); Non_hodgkin lymphoma and Hodgkin's disease (lymthoma); Solid tumor comprises breast cancer, lung cancer, oophoroma, carcinoma of testis, prostate cancer, colon cancer, melanoma, clear-cell carcinoma, neuroblastoma and neck tumour. Infected cell comprises by the cell of following any infected by microbes: bacterium, fungi, parasite or other any pathogenic microorganism.

" pathogen " is defined as any medium of containing nucleic acid and can causing the mankind, other mammal or vertebrate disease. The example of pathogen comprises bacterium, virus, protozoan, fungi, yeast, mould and the mycoplasma that causes the mankind, other mammal or vertebrate disease. The inhereditary material of pathogen can be DNA or RNA, and inhereditary material can be used as strand or double-strandednucleic acid exists. Pathogen may be positioned at outside the cell, or resides in the cell, such as the institute of the HIV in macrophage illustration.

Term donor leucocyte " infusion " and " transferring " are used convertibly at this, refer to identical method.

" light dosage " or " PCT dosage " is with J/cm²Unit determine, refer to total luminous energy in the per unit zone that the leucocyte sample is accepted in the PCT process. The time that is exposed to light by changing light intensity and cell sample can change light dosage.

With mW/cm²The light " intensity " that unit measures refers to the luminous energy that per second per unit sample is accepted.

" but photoactivation part " is defined as a kind of part that stands chemical modification with electromagnetic radiation at this. But when containing photoactivation compound partly by the electromagnetic radiation photoactivation, photoactivation part and nucleic acid form covalent bond, and form a kind of compound: nucleic acid complex is referred to herein as " adduct ".

" aromatics insert " refers to a kind ofly have aromatic ring structure, can insert compound or its part in the nucleic acid. The aromatics insert includes but not limited to anthracene, acridine, naphthalene, naphthoic acid, etc.

" cell colony of separation " is included in the cell colony that exists outside the normal resident biology of cell as used herein.

" immunologic competence " refers to immune cell such as T cell, B cell, NK cell, antigen presenting cell (APC) and the expressed function of other cell as used herein, and these functions include but not limited to: the processing of synthetic and secretion, antigen and the antigen fragment of the synthetic and secretion of cell factor, cell-mediated cytotoxicity, antibody and present, represent the expression of the surface markers of immunocyte feature. Be incorporated herein by reference

The reference of quoting among the application comprises being hereby incorporated by patent application and other publication of patent, announcement.Preferred embodiment is described

Below summed up a plurality of preferred aspect of the present invention, and in subsequently detailed description and embodiment, further described and illustrate.

The invention provides a kind of method, be used to handle isolating leukocyte population, can cause the degree of minimum GVHD, can effectively promote disease cell or the enough immunologic functions of pathogenic agent destructive but still keep so that the part of this colony can not be bred to this colony." minimum GVHD " meaning is that the degree of GVHD is not enough to cause mammiferous death, or eliminates the ability that leukocyte population promotes disease cell or pathogenic agent destruction and/or mediation GVL effect.

GVHD clearly with the clone of donor T cell expand relevant, in principle, removal, deactivation or to kill and wound leukocytic any processing all be effective to it.On the other hand, GVL is relevant with the leukocytic immunne response of donor, and may be the result of the synthetic or above combination of cytolysis, antigen presentation, the specific cell factor.To need leukocytic some function of donor be complete for observing the GVL effect.In the trial of avoiding GVHD, also make donor BM or white corpuscle can not be effectively provide necessary immunologic function for non-responsiveness person or BMT host to the deactivation of donor leukocyte function.

Immunoprotection still is provided and/or induces the GVL effect in order to make processing in the allogeneic host, eliminate GVHD, must optionally remove leukocytic multiplication capacity, and keep the leukocyte function of certain level.The invention provides leukocyte population with these features.

Many advantages of the colony of the use of addressing before isolating leukocyte population of the present invention provides and has been better than.A tangible advantage is that the morbidity of eliminating GVHD will allow the safety input of donor white corpuscle in the patient.Therefore, lack immunity system or have chimeric immune host and can accept heavy dose of treated donor white corpuscle and/or DLI repeatedly, thereby improved the usefulness of disease treatment.In the past, the threat of GVHD had limited the leukocytic amount of input.

The non-proliferative leukocyte population can provide the abundant immune defense of antagonism cancer and infection effectively for the host.These white corpuscles can be used for treating the hematologic malignancies of recurrence, as the leukemia and the myelomatosis of chronic marrow, acute myeloid, acute lymphocytic, multiple myeloma and other form, as a part for the treatment of behind the BMT or as a kind of selection method that is equipped with of BMT.The GVL effect that DLI causes not only can be used for removing minimum remaining disease, and can be used for the treatment of higher malignant cell load, especially because the shortage of white corpuscle expansion allows the safety input of greater amount cell.Treated leukocyte population of the present invention also can be used for the treatment of some solid tumor (for example mammary cancer and renal cell carcinoma) to the immune modulating treatment sensitivity, and can be used for preventing mating and mispairing allosome BMT process in transplant rejection.Other various clinical indication that this treated white corpuscle is suitable in treatment below has been described in detail in detail.

The leukocytic method of processing of the present invention comprises the following steps.Can from marrow, bleeding of the umbilicus or whole blood, obtain white corpuscle.The white corpuscle source of most convenient is a peripheral blood.Described in the scientific literature from other source and separated leukocytic method.For example, come purifying and enrichment white corpuscle by red corpuscle removal or white corpuscle electrophoretic process whole blood.The purifying leukocyte population about 99% that obtains does not contain red corpuscle, and comparing red corpuscle with white corpuscle has different sizes and density, is easy to remove by these methods.

Then with leukocyte population and compound, and do not damage effectively suppressing white corpuscle propagation under the condition of cell survival and integrity and handle.Remove unreacted compound, perhaps prolong this compound in time and can be changed into non-activity, and unnecessary removal.In a kind of preferred embodiment, " compound " is meant the compound that can form covalent linkage with a kind of nucleic acid.Covalent linkage be formed on per 10 ⁸Can form about 1-10 in the individual white corpuscle genomic dna base pair ⁴Individual adducts, preferably 5-10 ³Individual adducts, most preferably 10 ³Individual adducts.Importantly, the feasible non-specific damage to protein and other cellular constituent of treatment condition reduces to minimum and avoids membrane damage.Most of treated leukocytic film integralities are necessary for the GVL effect takes place.These treatment condition can not cause GVHD after also making treated white corpuscle effectively in introducing the allogeneic host, perhaps make it anergy in the MLR in vitro test.In addition, treated white corpuscle should keep for finish immunologic function, especially for promote disease cell or pathogenic agent in vivo the destruction effective protein proteins matter of (or external) express.

Following discloses can be with nucleic acid, preferably double-stranded DNA forms the character of the suitable compound of covalent linkage.These compounds can contain a kind of part that can form covalent linkage with nucleic acid, but this part is photoactivation or chemically reactive.This compound is by forming covalent linkage with genomic dna and then disturbing the function of archaeal dna polymerase and white corpuscle can not be bred, the propagation that suppresses cell can be regulated the quantity of the covalency adducts that compound and white corpuscle nucleic acid forms, make this compound can suppress propagation, but keep promoting the effective immunologic function of destruction of disease cell or pathogenic agent.Use the alkylating agent compound,, can regulate this effect by regulating compound concentration and white corpuscle contacts with compound before removing unconjugated compound time span.Required concentration depends on the character of specific compound, as solubleness in the aqueous solution and DNA binding constant.Generally with per 10 ⁸Can effectively produce about 1-10 in the individual white corpuscle genomic dna base pair ⁴The adducts of individual covalent attachment compound, about 5-10 preferably ³Individual adducts, more preferably about 10 ²-10 ³The concentration of individual adducts is used this compound.Ideally, handle the condition of leukocyte population per 10 ⁸To produce about 10 in the individual genomic dna base pair ³Individual adducts.The minimum compound concentration that can effectively obtain leukocyte compositions of the present invention is preferred concentration.

But use light-activated compounds, can regulate the PCT effect by the time span of regulating compound concentration, wavelength of light and being exposed to light.Use psoralene and other light-activated compounds to carry out the condition of PCT but below described in detail.

The purpose of treatment condition is per 10 ⁸Produce about 1-10 in the individual white corpuscle genomic dna base pair ⁴The adducts of individual covalent attachment compound, about 5-10 preferably ⁴Individual adducts, more preferably about 10 ²-10 ³Individual adducts.Ideally, these treatment condition are per 10 ⁸To produce about 10 in the individual genomic dna base pair ³Individual adducts.For example, by using radiolabeled DNA binding compounds as described in the following Examples, energy measurement is by the quantity of handling the compound-dna adduct that produces.

The compound of the another kind of type that can use in white corpuscle is handled is a kind of small molecules as the dna replication dna inhibitor.As mentioned above, this small molecules replication inhibitors can randomly contain a kind of linker (brittle or opposite) and a kind of effector part.Any step of dna replication dna can both be as the target that suppresses, and the formation, ORC that comprises origin recognition complex (ORC) is to initial, the extension that replenishes, duplicates of starting point etc.In addition, helping the inhibitor of the enzyme of reproduction process, as helicase and topoisomerase, also is useful.The example of topoisomerase enzyme inhibitor comprises, for example camptothecine and daunomycin.

In order to make white corpuscle sample and compound, compound of the present invention can be introduced in the leukocyte suspension (white corpuscle sample) with several forms.Can be used as the aqueous solution in water, the salt solution, synthetic medium (as " Sterilyte ^TM3.0 ") or the leukocytic solution introducing compound that suspends.Below provide and be suitable for the leukocytic solution of resuspension (blood transfusion level and non-blood transfusion level)." synthetic medium " is defined as water-based dextran or blood products storage medium at this.Can provide compound with the drying agent that contains or do not contain adjuvant in addition.Stir cell suspension then and mix this compound.

The white corpuscle of stand-by compound treatment is resuspended in the physiological balanced solution, as blood plasma, synthetic medium or its composition.Can provide white corpuscle with the volume of 200mL-1L.The white corpuscle that is used for handling preferably is contained in a kind of reaction vessel such as blood bag.The blood bag is known in the art.

Can monitor with the influence of compound treatment by external and in vivo test, thereby determine to make propagation and GVHD activity to reduce to minimum and make cytotoxicity function and/or GVL effect reach maximum optimal treatment condition leukocyte population viability and function.Top condition will be different with the character of compound used therefor, and be confirmed by disease or pathogenic agent.But for light-activated compounds, most preferred PCT condition comprises minimum compound concentration and minimum light dosage, and it is enough to provide propagation to be suppressed but can effectively promotes disease cell or pathogenic agent destructive leukocyte population.The leukocyte population that following sign is treated.

The cell survival of treated leukocyte population will be estimated.With as described in the lower section, for example, detect the sequence special as present embodiment, can measure the viability of cell in vivo the donor white corpuscle by pcr analysis.Preferably, this leukocyte population has the cell survival at least 3 weeks.Handle the leukocytic quick removing in back and can influence the ability that it induces the leukemia reaction.Processed leukocytic film integrality also is necessary for the GVL effect takes place.Can measure film integrality by trypan blue or the iodate third ingot dye excretion.If use,, optimize the PCT method in that the leukocyte count with intact cell film is reached aspect the maximum light dosage and/or compound concentration.

As described in following present embodiment, for example, can measure proliferation activity by limiting dilution test (LDA) or mixed lymphocyte reacion (MLR) test.The processed white corpuscle of the active indication of white corpuscle in mixed lymphocyte reacion (MLR) test mediates the ability of GVHD in vivo.In addition, GVHD symptom and/or the GVHD inductive M ﹠ M of having imported in the treated leukocytic MHC coupling animal by monitoring can be determined GVHD.

, the expression of surface antigen mark synthetic by the monitoring cytokine and the ability of dissolving target cell can be determined the white corpuscle activity.Utilize the expression of suitable antigen-specific antibodies and standard FACS-SCAN assay determination surface antigen mark.As the existence of function mensuration antigenicity mark CD2, CD28, CTLA4, CD40 part (gp39), CD18, CD25, CD69 (lymphocyte activator mark) and the CD16/CD56 of covalent attachment compound concentration and when using (if) PCT light dosage, known they participations interaction and immunologic function relevant with the NK cell-stimulating with the T cell.If PCT to the protein action temperature and, the stability prediction of PCT condition lower surface molecule is unaffected.Select for example PCT of treatment condition, make them can not influence the expression of surface molecular on the contrary or production of cytokines is reduced under the level of hope (above disclosed preferred levels).

For example, exist ⁵¹By the dissolving of people or L-1210, can measure the active dissolved cell activity of indication GVL in the Cr release test.As people such as Choudhury, blood 89:1133-1142,1997 described can be in external test leukemia effect, perhaps as people such as Johnson, blood 85:3302-3312,1995 describedly measure in vivo.The activity energy use-case of solid tumor such as the mouse model of solid tumor are detected.Mediation infected cell or pathogenic agent dissolved ability can be measured with infected animals in animal model.GVHD and GVL ability also can be measured as described in following examples 4 and 5 in vivo.

All above-mentioned tests have been described in following examples.Because the similarity of immunne response between mouse and the people, what the experiment of using mouse model to carry out was leted others have a look in advance replys.For example, the GVL effect all obtains fine proof in mouse and robot system.The result of these tests indicates biologically in the body among the host that DLI handles.

According to the test-results of animal model, the patient of representative quantity tested determine effective compound concentration, and use the active compound concentration that causes that patient disease alleviates.The condition that Most patients is used is most preferred.These patients generally are the patients after BM transplants.

According to the result of these tests and detection, determine that optimal treatment condition is possible, this condition will produce can not breed and cause GVHD, but can promote disease cell or pathogenic agent destructive leukocyte population effectively.Processed leukocyte population should have the GVL activity.The selection of white corpuscle donor

For DLI, host's (acceptor) of donor and white corpuscle infusion must be allochthonous.HLA-A and-B is a HLA I gene, and HLA-DR is a HLA II gene.Each of these genes all exists with multiple different allelic form.Because the karyomit(e) 6 (containing the HLA gene) of each individual intrinsic two copy, thus individual physical efficiency express the different HLA-A that reach as high as 6 ,-B and-DR protein (two not homoallelic products of each locus).Preferably, the allogeneic donor 3 of 6 HLA locus of HLA-A, B and DR or more on be that genotype is identical.In " monoploid is identical " transplanted, donor and host were mated on 3 of 6 HLA locus.Ideally, donor and acceptor are identical on HLA-A, B and DRB1.Can search the irrelevant donor of HLA-coupling by American National bone marrow donor program (NMPD).

Current, reach 55 years old patient for what the non-T cell of accepting unmodified get rid of to be transplanted, the patient is necessary identical with HLA-A, B and the DRB1 gene of donor.36 years old or younger patient can from HLA-A ,-B or-DR differs the donor that is no more than 1 minor antigen coupling and transplants.The less important coupling of HLA-A or B is defined as belonging to two kinds of antigens of identical cross reaction group.The less important coupling of HLA-DR is defined as expressing identical DR specificity but two kinds of different haplotypes of DRB1 allelotrope.For the scheme of suitable allogeneic donor, referring to O ' Reilly, people such as R., simplified marrow transplanting: be used to lack the patient's of donor method, in " hematology "-1996, the educational plan of U.S. hematology association, 132-146 page or leaf.Leukocytic preparation

The parent material of leukocyte population of the present invention can be provided by for example provincialism blood treatment center, and it is similar to the center in other source (as marrow) of existing processing peripheral hematopoietic stem cells and hemopoietic stem cell.Can and handle the white corpuscle of donor in any suitable facility input that is used to handle (can comprise that local Blood Center or hospital blood bank import) at multiple facility.In addition, the white corpuscle of the input cell treatment facility that can all through the night transport Good Manufacturing Practice (GMP) to is handled and quality assurance test.The treated white corpuscle that will be used for DLI then is sent to patient place hospital by express delivery on the same day, is used for the patient is used.In addition, the white corpuscle that can use method known in the art refrigeration to handle, as be controlled at chilling rate among the 10%DMSO, and be stored in (referring to people such as Russel, bone marrow transplantation 19:861-866,1997) in the liquid nitrogen.When needs are transplanted, the white corpuscle of melting chilling, DMSO is removed in washing.

A kind of ordinary method that is used for purifying white corpuscle preparation comprises by density gradient centrifugation separates from whole blood.This generally comprises the white corpuscle that is rich in the T cell by the separation of Ficoll gradient centrifugation.Referring to, for example, Longley and Stewart, immunological method magazine (J.Immunol.Methods) 121:33-38,1989.This method is following carries out: the blood sample (for example 50-200 milliliter) that (1) is carefully extracted at Ficoll upper strata Jia Xin makes the interface interference-free; (2) the opaque cell band (centrifugal back) that is positioned at gradient interface by sucking-off is collected white corpuscle; (3) cell of washing collection makes it not contain Ficoll.Precipitation red corpuscle and granulocyte in this system.In order to remove Ficoll, generally by centrifugal and abandon supernatant liquor, washed cell one or many.This method produces the white corpuscle preparation of purifying, promptly is substantially free of erythrocytic preparation.

The automatic mode that has a large amount of white corpuscle preparations of preparation.For example, the present invention has conceived according to working instructions white corpuscle electrophoresis apparatus (for example, COBE Spectra Apheresis System, COBE BCT, Inc.Lakewood, CO) processing blood.Preferably, use input (apheresis) instrument that produces white corpuscle preparation with minimum percentage hematocrit.Behind the white corpuscle electrophoresis,, make it to reach and be lower than 0.5% final hematocrit with suitable solution (aforesaid blood plasma, blood transfusion level solution etc.) washing and dilution white corpuscle preparation.If above step can not fully reduce hematocrit levels, then separate as further purification step with Percoll.As the electrophoretic a kind of selection method that is equipped with of white corpuscle, it is well-known for those skilled in the art that red corpuscle is removed method.

If it is desirable only containing the leukocyte population of T cell and NK cell or other white corpuscle mixture basically, for example then can utilize and select with the positive and/or negative of the suitable antibody of identifying the specific cell surface markers, by fluorescence-activated cell sorting method (FACS), from the white corpuscle preparation, separate selected cell subsets.The example of the surface markers that can be used to screen includes but not limited to CD4, CD8, CD16 and CD56.These cytopheresises are well known in the art, referring to, for example, Ed Harlow and David Lane, " antibody: laboratory manual " (Antibody, A Laboratory Manual), cold spring harbor laboratory, cold spring port, New York, 1988.

The leukocyte population of purifying is suspended in the as above disclosed suitable solution.Can handle the leukocyte population of purifying immediately, or refrigeration is used for later processing.In preparation, purifying, blended white corpuscle preparation are resuspended in isotonic solution such as blood plasma, synthetic medium or this both composition for the propagation deactivation.Generally with every milliliter of 10-10 ⁹Individual cell, preferably 10 ⁴-10 ⁷Cell/ml, ideally 2 * 10 ⁶The cell density of cell/ml prepares the leukocyte population of purifying.

The present invention has conceived treated leukocytic preparation and the multiple scheme of using or using.Can collect peripheral blood (or other white corpuscle source comprises marrow or bleeding of the umbilicus), and freezing up to being used to breed inactivation treatment, and thawing cell and washing make it not contain the refrigeration agent during processing, randomly process the white corpuscle preparation with the acquisition purifying, and handle.Subsequently can be with treated leukocyte population input patient.The following scheme of selecting fully also is possible: the i) collection of peripheral blood, processing, white corpuscle are handled and infusion; Ii) collect, processing, white corpuscle are handled, treated cell freezing, wash refrigeration agent and infusion off; Iii) inoculate donor to expand T cells with antigenic specificity, to collect peripheral blood, processing, processing and infusion from the donor of inoculation with target antigen; Iv) the external sensitization of donor white corpuscle is handled and infusion with expansion T cells with antigenic specificity, white corpuscle with target antigen.Compound

In one embodiment of the present invention, can be suitable for obtaining the purpose of leukocyte population of the present invention with the compound that nucleic acid forms covalent linkage.These compounds preferably comprise a kind of and the non-covalent bonded part of nucleic acid, and the identical or different part that can form covalent linkage with nucleic acid reaction.This compound preferably has following character: i) to the high binding affinity of nucleic acid; The ii) solubility in the aqueous solution; Iii) penetrate the ability of white corpuscle film.Can comprise the chemical reactivity part with the part that nucleic acid forms covalent linkage, but its do not need outside stimulus to activate and can with the part of nucleic acid reaction and photoactivation, its after stimulating with the electromagnetic radiation form just with nucleic acid reaction.

When this used, term " alkylating agent compound " was meant a kind of compound, and it comprises at least a chemical reactivity part, and this part can not form covalent linkage with nucleic acid reaction when having outside stimulus such as light stimulus.

But the compound that can form covalent linkage with nucleic acid also may be photoactivation." but light-activated compounds " in this definition comprises a kind of part that can form covalent linkage after being stung laser activation by the light of certain wavelength with nucleic acid.

Preferably, this compound comprise can with nucleic acid reaction form covalent linkage part and can with the non-covalent bonded part of nucleic acid.Within the scope of the invention, can with nucleic acid bonded part also can be used as can with the part of nucleic acid covalent reaction, but and can be for example photoactivation.

In one embodiment, the alkylating agent compound comprises: nucleic acid binding moiety; Can form the part (" being referred to herein as the effector part ") of covalent linkage with nucleic acid reaction; Fragility linker with covalently bound nucleic acid moiety and effector part.In a kind of preferred embodiment, in the aqueous solution, during proper pH value, these compounds have the activity of for some time, and they can combine with nucleic acid and reaction therebetween.After this stage, compound decomposition is for no longer can combine the product that can not react with it well with nucleic acid.

The chemical structure of these alkylating agent compounds can generally be described as a kind of can with the anchor of fragility linker covalent bonding, be covalently attached on a kind of effector." anchor ", be also referred to as " nucleic acid binding moiety " be defined as can with the non-covalent bonded part of biological nucleic acid polymer (DNA, RNA or its synthetic analogues)." effector " or " effector part " be defined as a kind of can be by forming the mechanism of covalent linkage and the part of this nucleic acid reaction with nucleic acid." fragility linker " is defined as a kind of part that is used for covalently bound anchor and effector, and it is degraded under certain conditions, makes anchor and effector no longer covalently bound.The arrangement of anchor-fragility linker-effector makes compound can combine (because binding ability of anchor) with nucleic acid specificity.This makes effector enter neighbouring and nucleic acid reaction.

Preferably, nucleic acid binding moiety is selected from: aromatics insert, acridine, acridine derivatives, ellipticine, 2-polyamines, ditch wedding agent and hydrophobic or select the shape wedding agent.In preferred embodiments, the fragility linker contains a kind of functional unit, and it is selected from: forward ester, reverse ester, forward monothioester, reverse monothioester, forward and reverse thion acid esters, forward and reverse dithionic acid, sulfuric ester, forward and reverse sulphonate, phosphoric acid ester and forward and reverse phosphonate group.The effector part preferably contains a kind of functional group, is selected from: mustard seed base, the suitable thing of mustard seed base, epoxide, aldehyde and formaldehyde compound body.

When alkylating agent compound and leukocyte compositions combine a kind of reaction mixture of formation under physiological pH, the effector part of this compound and the nucleic acid reaction that is contacted.Can not with the effector of nucleic acid reaction part gradually by the solvent hydrolysis.The hydrolysis of fragility linker and effector-nucleic acid reaction and effector hydrolysis take place simultaneously.It is desirable to the fragility linker with low as to be enough to make the speed of white corpuscle propagation inactivation to be decomposed; That is, the rate of decomposition of fragility linker is lower than the speed of this compound and white corpuscle nucleic acid reaction.Through after the sufficiently long time, this compound decomposition is anchor (it also can have fragility linker fragment) and effector-nucleic acid degradation production (fragility linker fragment also may still be connected with effector), perhaps is decomposed into the effector degradation production (fragility linker fragment also may still be connected with effector) of anchor (it also can have fragility linker fragment) and hydrolysis.Not with degradation that anchor or effector keep being connected after, also can produce other fragment of fragility linker.Alkylating agent compound of the present invention is executed scheme really conscientiously and is determined whether anchor degradation production or effector degradation production have fragility linker fragment, perhaps whether produce other fragment of fragility linker, and these fragments do not combine with anchor or effector degradation production.

Preferred alkylating agent compound is the compound that produces low mutagenicity degradation production in fragility linker division back.The mutagenicity of compound is mainly owing to the anchor part after the effector hydrolysis, because this anchor and nucleic acid interaction, and may have the ability that interfere RNA is duplicated, even still like this after the effector partial hydrolysis.After the division of fragility linker, the anchor fragment has the mutagenicity that reduces greatly.

In other embodiments of the present invention, from reaction mixture, remove the compound that can form covalent linkage over time with nucleic acid.Optimum reacting time can be determined to experience, described in hereinafter embodiment.The U.S. Patent Application Serial of owning together 08/779,830,08/779,885 and 09/003, in 113 and in the U.S. Patent application proxy catalog number (Cat.No.) 28217-20004.21 that owns together and 28217-20004.22 of application on July 8th, 1998, provide the method and composition that is used for removing compound from reaction mixture.The disclosure of last all mentioned patent applications all is incorporated herein by reference fully at this.In addition, also in reaction mixture, add the quencher that can react and make it inactivation with compound.In the U.S. Patent application proxy catalog number (Cat.No.) 28217-20006.00 of the U.S. Provisional Patent Application series number of owning together 60/070,597 of on January 6th, 1998 application and application on July 6th, 1998, typical quencher and method are disclosed; Its disclosure all is incorporated herein by reference fully at this.

Broad variety is applicable to as anchor, linker and effector.The example of the anchor group that can use in the alkylating agent compound includes but not limited to: insert (comprising the aromatics insert), minor groove binding, major groove wedding agent, by electrostatic interaction or hydrophobic interaction bonded molecule and by sequence-specific interaction bonded molecule.Below be the non-limiting tabulation of possible anchor group:

Acridine (and acridine derivatives, proflavine for example, acriflavine, two acridines, dihydroketoacridine, benzacridine, quinacrine), actinomycin, anthracene nucleus ketone, rhodomycetin, daunomycin, thioxanthone (and thioxanthone derivates, romicil D for example), anthramycin, mitomycin, Quinomycin A (levomycin), triostin, ellipticine (and dimer, tripolymer and analogue thereof), norphilin A, fluorenes (and derivative, as Fluorenone, fluorenediamine), azophenlyene, phenanthridines, thiodiphenylamine (for example chlorpromazine) phenoxazine, benzothiazole, xanthene and thioxanthene, anthraquinone, the anthracene pyrazoles, the benzo thiapyran indoles, 3, the 4-benzopyrene, 1-pyrenyl oxyethane, benzanthrene, benzodipyrane ketone, quinoline (chloroquine for example, quinine, phenylquinoline, carboxamide), furocoumarin(e) (for example psoralene and isopsoralen), the second ingot, third ingot, coralyne and polynuclear aromatics and epoxyethane derivative thereof;

Distamycin, spindle mycin, other lexitropsin, Hoechst 33258 and other Hoechst dyestuff, DAPI (4 ', 6-diamidino-2-phenylindone), berenil and triarylmethane dye;

Aflatoxin;

Spermine, spermidine and other polyamines; With

Nucleic acid or analogue, they interact by sequence-specific and form and combine with the direct base pairing of strand target as triple helix formation, D-ring.The derivative of these compounds also is the limiting examples of anchorage, and wherein compound derivatives includes but not limited to, at any band of position the substituent compound of one or more any kind ofs is arranged, this compound oxidation or reduzate, or the like.

The example of the linker that can use in the present invention includes but not limited to contain the compound of following functional group, and (wherein the carbonyl carbon of this ester is positioned at the sp of anchor and ester as ester ³Between the oxygen; This arrangement also is called " forward ester "), " oppositely ester " (sp of this ester wherein ³Oxygen is between the carbonyl carbon of anchor and this ester), (wherein the carbonyl carbon of this monothioester is between the sulphur of anchor and this monothioester for monothioester, also be called " forward monothioester "), oppositely monothioester (wherein the sulphur of this monothioester also is called " reverse monothioester " between the carbonyl carbon of anchor and this monothioester), forward and reverse thion acid esters, forward and reverse dithionic acid, sulfuric ester, forward and reverse sulphonate, phosphoric acid ester, forward and reverse phosphonate group." monothioester " expression-C (=O)-the S-base; " thion acid esters " expression-C (=S)-the O-base, " dithionic acid " expression-C (=S)-the S-base.For the group that is called as " forward " and " oppositely ", forward is such functional group's direction, and wherein after functional group's hydrolysis, the acidic functionality of generation can be covalently bound with the anchor part, and the alcohol or the thiol functionalities that produce can be covalently bound with the effector part.Oppositely be such functional group's direction, wherein after functional group's hydrolysis, the acidic functionality of generation can be covalently bound with the effector part, and the alcohol or the thiol functionalities that produce can be covalently bound with the anchor part.

The example of the effector that can use in the present invention includes but not limited to: mustard seed base, the suitable thing of mustard seed base, epoxide, aldehyde, formaldehyde compound body and other alkanisation and linking agent.The mustard seed base is defined as containing single or two halogen ethylamino-and single halogen ethyl-sulfide base.The suitable thing of mustard seed base be defined as by being similar to the mustard seed base mechanism (promptly; by forming a kind of ethylene imine intermediate; perhaps by containing or forming a kind of aziridine ring; it can react with nucleophile) reaction group, as single or two methylsulfonyl ethylamino-, single methylsulfonyl ethyl-sulfide base, list or two tosyl group ethylamino-and toluene monooxygenase alkylsulfonyl ethyl-sulfide base.The formaldehyde compound body is defined as being decomposed into any compound of formaldehyde in the aqueous solution, comprise hydroxyl methylamine such as methylol glycine.U.S. Patent number 4,337,269 and International Patent Application WO 97/02028 in provided the example of formaldehyde compound body.

Can be used for preparing leukocytic alkylating agent compound of the present invention with following general formula I, II and III description.

General formula I is:

R at least wherein ₁, R ₂, R ₃, R ₄, R ₅, R ₆, R ₇, R ₈And R ₉One of be as described below-V-W-X-E, and R ₁, R ₂, R ₃, R ₄, R ₅, R ₆, R ₇, R ₈And R ₉Residue person be independently selected from :-H ,-R ₁₀,-O-R ₁₀,-NO ₂,-NH ₂,-NH-R ₁₀,-N (R ₁₀) ₂,-F ,-Cl ,-Br ,-I ,-C (=O) R ₁₀,-C (=O)-O-R ₁₀With-O-C (=O)-R ₁₀,

Wherein-R ₁₀Be independently: H ,-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

V is-R independently ₁₁-,-NH-R ₁₁-or-N (CH ₃)-R ₁₁-, wherein-R ₁₁-be independently :-C _1-8Alkyl-,-C _1-8Assorted alkyl-,-aryl-,-heteroaryl-,-C _1-3Alkyl-aryl-,-C _1-3Assorted alkyl-aryl-,-C _1-3Alkyl-heteroaryl-,-C _1-3Assorted alkyl-heteroaryl-,-aryl-C _1-3Alkyl-,-aryl-C _1-3Assorted alkyl-,-heteroaryl-C _1-3Alkyl-,-heteroaryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-aryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl-or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl-;

W is independently-C (=O)-O-,-O-C (=O)-,-C (=S)-O-,-O-C (=S)-,-C (=S)-S-,-S-C (=S)-,-C (=O)-S-,-S-C (=O)-,-O-S (=O) ₂-O-,-S (=O) ₂-O-,-O-S (=O) ₂-,-O-P (=O) (OR ₁₀)-O-,-P (=O) (OR ₁₀)-O-,-O-P (=O) (OR ₁₀)-;

X is-R independently ₁₁-; And

E is independently selected from :-N (R ₁₂) ₂,-N (R ₁₂) (R ₁₃) ,-S-R ₁₂With

Wherein-R ₁₂For-CH ₂CH ₂-G, wherein each G be independently-Cl ,-Br ,-I ,-O-S (=O) ₂-CH ₃,-O-S (=O) ₂-CH ₂-C ₆H ₅Or-O-S (=O) ₂-C ₆H ₄-CH ₃

R wherein ₁₃Be independently :-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

And all salt and steric isomer (comprising enantiomer and diastereomer).

A kind of preferred composition according to general formula I is a compound S-303, and wherein V is-NHR ₁₁-, R ₁₁For-CH ₂CH ₂-, W is-C (=O)-and O-, X is-CH ₂CH ₂-, E is-N (R ₁₂) ₂, R ₁₂For-CH ₂CH ₂G, G is-Cl.Referring to the PCT application US98/00531 that owns together.General formula I I is:

R wherein ₁, R ₂, R ₃, R ₄, R ₅, R ₆, R ₇And R ₈Be independently selected from :-H ,-R ₁₀,-O-R ₁₀,-NO ₂,-NH ₂,-NH-R ₁₀,-N (R ₁₀) ₂,-F ,-Cl ,-Br ,-I ,-C (=O) R ₁₀,-C (=O)-O-R ₁₀With-O-C (=O)-R ₁₀,

Wherein-R ₁₀Be independently: H ,-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

R ₂₀For-H or-CH ₃And

R ₂₁For-R ₁₁-W-X-E,

Wherein-R ₁₁-be independently :-C _1-8Alkyl-,-C _1-8Assorted alkyl-,-aryl-,-heteroaryl-,-C _1-3Alkyl-aryl-,-C _1-3Assorted alkyl-aryl-,-C _1-3Alkyl-heteroaryl-,-C _1-3Assorted alkyl-heteroaryl-,-aryl-C _1-3Alkyl-,-aryl-C _1-3Assorted alkyl-,-heteroaryl-C _1-3Alkyl-,-heteroaryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-aryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl-or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl-;

W is independently-C (=O)-O-,-O-C (=O)-,-C (=S)-O-,-O-C (=S)-,-C (=S)-S-,-S-C (=S)-,-C (=O)-S-,-S-C (=O)-,-O-S (=O) ₂-O-,-S (=O) ₂-O-,-O-S (=O) ₂-,-O-P (=O) (OR ₁₀)-O-,-P (=O) (OR ₁₀)-O-,-O-P (=O) (OR ₁₀)-;

X is-R independently ₁₁-; And

E is independently selected from :-N (R ₁₂) ₂,-N (R ₁₂) (R ₁₃) ,-S-R ₁₂With

Wherein-R ₁₂For-CH ₂CH ₂-G, wherein each G be independently-Cl ,-Br ,-I ,-O-S (=O) ₂-CH ₃,-O-S (=O) ₂-CH ₂-C ₆H ₅Or-O-S (=O) ₂-C ₆H ₄-CH ₃

R wherein ₁₃Be independently :-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

And all salt and steric isomer (comprising enantiomer and diastereomer).

General formula III is:

R at least wherein ₄₄, R ₅₅, R ₃, R ₄, R ₅And R ₈One of be-V-W-X-E and R ₄₄, R ₅₅, R ₃, R ₄, R ₅And R ₈Residue person be independently selected from :-H ,-R ₁₀,-O-R ₁₀,-NO ₂,-NH ₂,-NH-R ₁₀,-N (R ₁₀) ₂,-F ,-Cl ,-Br ,-I ,-C (=O) R ₁₀,-C (=O)-O-R ₁₀With-O-C (=O)-R ₁₀,

Wherein-R ₁₀Be independently: H ,-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

V is-R independently ₁₁-,-NH-R ₁₁-or-N (CH ₃)-R ₁₁-, wherein-R ₁₁-be independently :-C _1-8Alkyl-,-C _1-8Assorted alkyl-,-aryl-,-heteroaryl-,-C _1-3Alkyl-aryl-,-C _1-3Assorted alkyl-aryl-,-C _1-3Alkyl-heteroaryl-,-C _1-3Assorted alkyl-heteroaryl-,-aryl-C _1-3Alkyl-,-aryl-C _1-3Assorted alkyl-,-heteroaryl-C _1-3Alkyl-,-heteroaryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-aryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl-,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl-,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl-or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl-;

W is independently-C (=O)-O-,-O-C (=O)-,-C (=S)-O-,-O-C (=S)-,-C (=S)-S-,-S-C (=S)-,-C (=O)-S-,-S-C (=O)-,-O-S (=O) ₂-O-,-S (=O) ₂-O-,-O-S (=O) ₂-,-O-P (=O) (OR ₁₀)-O-,-P (=O) (OR ₁₀)-O-,-O-P (=O) (OR ₁₀)-;

X is-R independently ₁₁-; And

E is independently selected from :-N (R ₁₂) ₂,-N (R ₁₂) (R ₁₃) ,-S-R ₁₂With

Wherein-R ₁₂For-CH ₂CH ₂-G, wherein each G be independently-Cl ,-Br ,-I ,-O-S (=O) ₂-CH ₃,-O-S (=O) ₂-CH ₂-C ₆H ₅Or-O-S (=O) ₂-C ₆H ₄-CH ₃

R wherein ₁₃Be independently :-C _1-8Alkyl ,-C _1-8Assorted alkyl ,-aryl ,-heteroaryl ,-C _1-3Alkyl-aryl ,-C _1-3Assorted alkyl-aryl ,-C _1-3Alkyl-heteroaryl ,-C _1-3Assorted alkyl-heteroaryl ,-aryl-C _1-3Alkyl ,-aryl-C _1-3Assorted alkyl ,-heteroaryl-C _1-3Alkyl ,-heteroaryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-aryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Assorted alkyl-heteroaryl-C _1-3Alkyl ,-C _1-3Assorted alkyl-aryl-C _1-3Assorted alkyl ,-C _1-3Alkyl-heteroaryl-C _1-3Assorted alkyl or-C _1-3Assorted alkyl-heteroaryl-C _1-3Assorted alkyl;

And all salt and steric isomer (comprising enantiomer and diastereomer).

Should be appreciated that in above-mentioned general formula I acridine nuclear is the anchor part ,-V-W-X-group comprises that fragility linker, E group are the effector group.Equally, in above-mentioned general formula III, psoralene nuclear is the anchor part, and-V-W-X-group comprises that fragility linker, E group are the effector group.General formula I I is the subclass of general formula I.

Embodiment 7-12 illustrates the synthetic of these compounds, is used to produce white corpuscle of the present invention.

In one embodiment of the present invention, the U.S. Patent number of owning together 5 in the past, 399, disclosed 4 '-(4-amino-2-oxa-) butyl-4 in 719 (its disclosure is incorporated herein by reference fully at this), 5 ', 8-trimethylpsoralen (" S-59 ") also can be used for the photochemical treatment to leukocyte population.

But the light-activated compounds that is applicable to the inventive method comprises: furocoumarin(e), actinomycin, anthracene nucleus ketone, anthramycin, benzodipyrane ketone, fluorenes and Fluorenone, single star fat indigo plant, norphillinA, organic dye, phenanthridines, thiodiphenylamine sulfosalt, azophenlyene, thiodiphenylamine, triazobenzene, quinoline and thioxanthone.But the preferred kind of light-activated compounds described herein typically refers to furocoumarin(e).Psoralene and derivative

Psoralene is the plane aromatic organic compounds that belongs to the furocoumarin(e) group.Psoralene is present in the nature, mainly in plant, comprises lime tree, cloves, celery, Selinum pastinaca and Fructus Fici.As for the mankind, psoralene has been used for the photochemotherapy to vitiligo, psoriasis and cutaneous T cell lymphoma treatment.About the photochemical summary of psoralene, referring to Parsons, B.J., photochemistry and photobiology 32:813-821,1980.The basic structure that has below shown psoralene.

Especially, the present invention considers psoralene, [the 7H-furans (3,2-g)-(1)-chromene-7-ketone, or the acrylic acid b-lactone of 6-hydroxyl-5-cumarone], it is linear:

Two oxygen residues that wherein are additional to the center aromatic portion have 1,3 direction, and furan nucleus wherein partly is connected on the position 6 of coumarin system of two rings in addition.3,4,5,8,4 ' or 5 ' replacement is produced the psoralene derivative in the position by linear furocoumarin(e).

About safety problem, psoralene has been present in our diet.When no long wavelength UVA shone, psoralene was relative non-activity.Be several milliseconds effective half-life that records UVA activated psoralene.Remaining all medicines in irradiation back are all replied and are the non-activity state, thereby limit its side effect.

Be used in the past pathogen inactivated scheme need be before being exposed to light and photoperiod between remove molecular oxygen from reaction, prevent the damage of the oxyradical that produces in the irradiation process to blood products.Referring to people such as L.Lin, blood 74:517 (1989); The U.S. Patent number 4,727,027 of Wiesehahn.Method of the present invention can be used for suppressing leukocytic propagation in the presence of oxygen.In addition, use the novel psoralene that method of the present invention is used, do not need to reduce the concentration of molecular oxygen.Preferred psoralene has low mutagenicity and high nucleic acid binding affinity.

8-methoxypsoralen (different titles is arranged in the literature, for example, xanthotoxin, xanthotoxin, 8-MOP) be a kind of naturally occurring psoralene, in Ames test, have low mutagenicity.

4 '-aminomethyl-4,5 ', 8-trimethylpsoralen (AMT) is that the nucleic acid of responding property most is in conjunction with one of psoralene derivative, can in per 3.5 DNA base pairs, produce and to reach 1 AMT adducts (S.T.Issacs, G.Wiesehahn and L.M.Hallick, NCI monograph (NCI Monograph) 66:21,1984).

" 4 '-primary amino replace psoralene " or " 4-PAP " is defined as such psoralene compound, and it has by length overall is the NH that the hydrocarbon chain of 2-20 carbon is connected with psoralene 4 ' position ₂Base, wherein the 0-6 of these carbon is replaced by NH or O independently, and other point of the every bit of replacement and replacement separates two carbon at least, separates a carbon at least with psoralene.The psoralene that 4 '-primary amino replaces can have other replacement on 4,5 ' and 8 positions of psoralene, these replacements include but not limited to following groups: H and (CH ₂) _nCH ₃, n=0-6 wherein.

" 5 '-primary amino replace psoralene " also with abbreviation " 5-PAP " expression, is defined as such psoralene compound at this, and it has by length overall is the NH that the hydrocarbon chain of 1-20 carbon is connected with psoralene 5 ' position ₂Base, wherein the 0-6 of these carbon is replaced by NH or O independently, and other point of the every bit of replacement and replacement separates two carbon at least, separates a carbon at least with psoralene.The psoralene that 5 '-primary amino replaces can have other replacement on psoralene 4,4 ' and 8 positions, these replacements include but not limited to following groups: H and (CH ₂) _nCH ₃, n=0-6 wherein.U.S. Patent number 5,585 discloses example (comprising S-59) and their synthetic method of 4-PAP and 5-PAP in 503,5,578,736,5,556,993 and 5,399,719.

Preferred psoralene comprises: psoralene (being commonly referred to as PAP at this), 8-methoxypsoralen (8-MOP), 4 '-aminomethyl-4 that the psoralene and 4 ' that 5 '-primary amino replaces-primary amino replaces, 5 ', 8-trimethylpsoralen (AMT), 5-MOP (5-MOP) and trioxa quinoline 4,5 ', the 8-trimethylpsoralen.AMT, 5-MOP, 8-MOP and trioxa quinoline can commercially obtain.

Most preferred psoralene be have above shown in the S-59 (see abbreviation S-59) of structural formula.S-59 is that a kind of energy is by embedding and the synthetic psoralene of the reversible bonded of nucleic acid.In case with UVA irradiation, the S-59 of embedding promptly form single adducts and with RNA and DNA interchain linkage.The S-59 photochemical treatment is a nucleic acid specificity, and S-59 facilitates penetration of cytolemma and nuclear membrane.S-59 has the characteristics of highly water-soluble and nucleic acid binding affinity concurrently, has high quantum yield for the formation of nucleic acid adducts.This makes low psoralene concentration can be used for PCT, suppresses propagation under the situation of no non-specific cell damage.The mutagenicity of S-59 and genotoxicity studies have shown that concentration 40000-67000 safety margin doubly for the pathogenic agent in the goods that are used for purifying the blood.At last, the S-59 dosage that is used for PCT is significantly less than average dietary intake every day (Wagstaff.D.J.Reg.Tox.Pharm.14:261-272,1991) of furocoumarin(e) (chemical group that comprises psoralene).

In one embodiment, but handle the cell colony that contains a kind of leukocyte population with a kind of light-activated compounds, this compound contains a kind of part that can form covalent linkage after photoactivation with nucleic acid.Therefore, in order to form covalent linkage with nucleic acid, but light-activated compounds must activate with light.Light comprises ultraviolet ray (UVA, UVB, UVC) and visible light.In one embodiment, the wavelength region that is used for PCT is 200-450nm.In a kind of preferred embodiment, the wavelength of light that is used for PCT is 320-400nm.

Preferably, with wavelength be this cell colony of UV linear light chemical treatment of 320-400nm.Simple UVA irradiation pair cell causes minimum damage.

In one embodiment, but light-activated compounds comprises a kind of psoralene molecule.The PCT that uses proper concn psoralene and UVA line dosage will cause that propagation suppresses, and keep leukocytic immunogenicity function, for example, this function can promote the destruction of disease cell, infected cell or pathogenic agent effectively, induce the leukemia reaction, be convenient to the implantation of second kind of cell colony, promote immunologic reconstitution, be beneficial to immunotherapy and treatment mixing chimerism.The psoralene reactivity not only especially at the nucleic acid in the cell in protein, and in the RNA molecule different psoralene binding affinities is arranged also for DAN, this is because the difference (Cimino of the nucleic acid construct in the feasible better intercalation of DNA, G.D. wait the people, state (Ann.Rev.Biochem.) 54:1151-1193 bioid academic year, 1985).In addition, utilize this technology, can modify transcriptional activity (comparing non-activity) gene in distinctiveness ground, this is owing to can influence psoralene bonded not isomorphic map and histone bonding state.Therefore, have the mainly ability of interference cell function on synthetic (duplicating) level of DNA,, protein synthesis is had minimum influence or do not have influence transcribing less influence as the processing of PCT.For example, in the situation of PCT, determine the interferential level according to used psoralene concentration and UVA line dosage.

But with light-activated compounds blended white corpuscle sample with a kind of photoactivation device photochemical treatment.Usually, be applicable to that the photoactivation device of this PCT method comprises following part: (a) a kind of device, be used to provide the electromagnetic radiation of suitable wavelength, but to cause the activation of light-activated compounds; (b) device of support several samples during photoactivation, it has the fixed relation with the radiating device is provided; (c) during photoactivation, make sample temperature remain in device in the temperature range of hope.

In one embodiment, this photoactivation device can be launched the electromagnetic radiation spectrum of certain strength, comprises 200-450nm, the wavelength of 320-400nm preferably.Use the suitable wave filter in the photoactivation device can select certain wavelengths.At people's such as Hearst U.S. Patent number 5,184,020 and 5,503, but 721 and WO 96/39820 in a kind of suitable photoactivation device and the method for utilizing this device photoactivation light-activated compounds are disclosed.Operable other photoactivation device comprises General Electric F20TI2-BLB type fluorescence UVA bulb (Alter, H.J. wait the people, lancet 24:1446 (1988)) and the A405-TLGW/05 type long wavelength ultraviolet lamp made of London P.W.Allen Co..With psoralene to leukocytic photochemical treatment

Preparation psoralene stock solution in water or in appropriate solvent as described in the following Examples.

The psoralene stock solution is added the final concentration of extremely wishing in the donor leukocyte population suspension of purifying.With above-mentioned preparation method, the PAS saturated with 8-MOP prepares the unit that handles with 8-MOP.In the photoactivation device, shine the donor white corpuscle of purifying, to 10 with the UVA line ^-3-100J/cm ²Whole dosage.Stirred sample between the light period.Before irradiation, take the white corpuscle sample in contrast.

Generally speaking, adding concentration in the white corpuscle preparation of purifying is 10 ^-4-150 μ M, preferably 10 ^-3The PAP of-150 μ M.Leukocytic cell density is generally about 10-10 ⁹Cell/ml, preferably about 10 ³-10 ⁸Cell/ml, more preferably about 10 ⁴-10 ⁷Cell/ml, most preferably about 2 * 10 ⁶Cell/ml,

In a kind of preferred embodiment, utilize wavelength for the ultraviolet ray of 320-400nm to carrying out PCT with S-59 blended white corpuscle.The wavelength that the photoactivation method is limited to greater than 320nm makes direct nucleic acid damaging reduce to minimum, because the absorption of nucleic acid is very little when being higher than 313nm.Cell preferably will accept about 10 ^-3-100J/cm ², 1-3J/cm more preferably ²Light dosage.In a kind of the most preferred embodiment, light dosage is 3J/cm ²Irradiation is generally 1-50mW/cm ²Light intensity.The time that the white corpuscle sample is exposed to the UV line is about 1 second to 60 minutes, preferably about 3 minutes, and more preferably about 1 minute.

Behind the PCT, as above reach the described mensuration white corpuscle of embodiment sample propagation and participate in the active usefulness of white corpuscle.Mainly be to measure following parameters: cell survival, multiplication capacity, cytokine secretion and surface antigen are expressed.Treatment is used

Treated leukocyte population of the present invention be do not breed but keep immunologic competence, as described below have multiple prevention and a therepic use.Usually, the individuality of accepting to contain the DLI of treated leukocyte population includes but not limited to the transplant patient, carries out the patient of solid organ transplantation as transplant patient behind the BM or expection.Treated white corpuscle can not cause that this fact of GVHD makes and can use more substantial white corpuscle (promptly bigger cell dosage) in DLI, thereby make the usefulness of treatment reach maximum.

Treated white corpuscle can be used for donor leukocyte infusion (DLI), especially provides immunocompetence to allosome BMT patient or non-responsiveness patient (as suffer from CLL gerontal patient).Use treated leukocytic DLI to can be used for alleviating and comprise following various disease conditions:

Leukemia comprises chronic lymphocytic leukemia (CML), lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CmML), acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL); Multiple myeloma (MM); Non_hodgkin lymphoma and He Jiejin lymphomas; Large celllymphoma (LCL); With Epstein-Barr virus inductive B lymphocytic hyperplasia disease (EBV-BLPD or EBV-inductive lymphoma);

Solid tumor comprises mammary cancer, lung cancer, ovarian cancer, carcinoma of testis, prostate cancer and colorectal carcinoma, melanoma, renal cell carcinoma, neuroblastoma, neck tumour;

Aplastic anemia; Myelodysplasia;

Immune deficiency, for example common panimmunity defective, SCID, Wei-Ao two syndromes;

Agranulocytosis;

Mix chimerism; With

Inherited disease.

DLI can be used to prevent or the various ways of therapeutic purpose:

(a) to the adoptive immunotherapy of all allotransplantations;

(b) for immunosuppression or immune deficiency patient's immunologic reconstitution;

(c) to the old leukaemic's that is unsuitable for transplanting treatment

(d) a small amount of or small-sized transplanting of BMT.

The a small amount of scheme of BMT is the method for two steps, comprising: i) use minimal condition scheme and stem cell donator inducing transplantation tolerance; Ii) repeat treatment with DLI.This scheme has been avoided the toxicity of chemotherapy scheme.The a small amount of scheme of BMT is applicable to following indication: can not normally be suitable for patient, the especially gerontal patient (＞65 years old) that transplant; CLL patient, the wherein serious non-responsiveness of great majority.The a small amount of scheme of BMT has reduced toxicity, GVHD and infection.The a small amount of scheme of a kind of typical B MT has been described in following examples 13.

(e) CD34 select/allotransplantation of T cells exclude adds DLI simultaneously.

The DLI that the cell of selecting with CD34+ (for the selection of stem cell enriched and progenitor cell) provides simultaneously can be used for providing the adoptive immunotherapy to infecting, the infection that causes as CMV, Epstein-Barr virus (EBV), adenovirus (Ad), kaposi sarcoma-associate herpesvirus, and fungi infestation, and can be used for treating leukemia, for example CML.

(f) treatment of mixing chimerism.

(g) remedy chemotherapy or chemoresistance leukemia.

In one embodiment of the present invention, in transplanting, monoploid identical (doubly single) imposes PA-DLI.Usually, single T cells exclude that needs almost completely of transplanting causes immunosuppression serious, that continue.PA-DLI can be used for keeping host's immunocompetence; In addition, can be treatment for cancer GVL is provided effect.

In preferred embodiments, no GVHD ability of the present invention but keep the white corpuscle of some immunologic function, removal, the especially CML and acute leukemia, lymphoma and the multiple myeloma that in DLI, are used for minimum remaining disease behind the alleviation of relapsed cancer or the allogeneic BMT.The usefulness that the special a large amount of donor CTL of the bcr-abl protein of CML is helped to recur DLI among the CML.Can detect malignant cell if be conventionally used for the method for monitoring cancer cells existence by the doctor, determine that then the cancer patients suffers from " recurrence ".

In another embodiment, during inducing chemotherapy (chemotherapeutical the junior one wheel), use in leukaemic's the treatment and contain the leukocytic DLI of non-proliferative, avoid severe bone marrow depression scheme and eliminate minimum remaining disease.In this arrangement, use DLI, can reduce the poisonous dosage of used chemotherapeutics between inductive phase.

In one embodiment of the present invention, PA-DLI also can be used for the transplanting (for example kidney, heart, skin) of solid organ, as a kind of method that improves the chance of allogeneic life-span and donor organ acceptance.Because the existence of passenger leukocyte in the donor organ, big multigenerator official repels in heteroplastic transplantation.Owing to not breeding and can not mediating GVHD, tolerance scheme before DLI (using the white corpuscle of organ donor) can be used as and transplants is used for and will otherwise induces the inhibition of organ rejection's immunne response.For the host's of solid organ transplantation tolerance comprise the following steps: before the organ transplantation process or during, contain or do not contain the PA-DLI input of stem cell donator with the suitable severe bone marrow depression scheme of level, donorcells can be survived in circulation.DLI can be convenient to the implantation of stem cell donator in this scheme, allows the mixing chimerism among the host.

In all types of transplanting and implantation situation, can be before transplanting, simultaneously or afterwards the host is used treated white corpuscle.Can carry out repeatedly the infusion of treated cell colony in any time in these times.

White corpuscle that do not breed, immunologic function is for avoid the patient's of traditional allosome BMT DLI particularly important owing to advanced age or comorbid are sick.The gerontal patient (＞65 years old) who suffers from cancer is not the main candidate of allosome BMT, partly is because transplanting can not be rebuild the aged immunity system well, and because the severity of this method.Current, be chemotherapy to the effective unique selection of cancer among the control gerontal patient, its side effect is intolerable for these patients.Treated white corpuscle of the present invention provides the selection method that is equipped with that a kind of ten minutes needs, and is used for the treatment of aged patient's cancer and immune defense to opportunistic infection is provided.DLI does not cause that for destruction of cancer cells the GVHD that follows is useful.

In some treatment situation, before with above-mentioned compound treatment donor leukocyte population, expansion donor white corpuscle subgroup especially T cells with antigenic specificity and precursory cell is desirable.Can use disease cell (for example, tumor associated antigen) or the special antigenic stimulation leukocyte population of pathogenic agent, expand/enrichment is to the cytotoxicity of this antigen-specific and the quantity of helper cell.By before being separated white corpuscle the donor that stimulates with suitable antigen inoculation donor, can stimulate in vivo; In addition, before with the compound treatment leukocyte population, can carry out separating leukocytic stimulation external.

For example, the donor with the IgG of multiple myeloma patients inoculation contains the special CTL of predecessor of high-caliber myelomatosis.Therefore, for the application in the treatment multiple myeloma (MM), the preparation white corpuscle is preferred from the donor of these inoculations.Hsu, people such as FJ, national medical science (Nat.Med.) 2:52-58, described in 1996 for the non_hodgkin lymphoma immune patients and with tumor-associated peptides to the dendritic cell pulse.At Stingl, people such as G., immunity today (Immunol.Today) 16:330-333 has summarized in 1995 with the technology of peptide/antigen to the dendritic cell pulse.Pardoll, people such as D., immunity (Immunity) 3:165-169 has described cDNA immunity (with suitable cDNA transfection dendritic cell) in 1995.At the adoptive immunotherapy that is used for the CML treatment, before separating the donor white corpuscle, can be used for by method treatment of the present invention and infusion subsequently with bcr-abl peptide vaccination donor.For example, ten Bosch, people such as G., blood 88:3522-3527,1996 have described the effective and immunogenic peptide that is used to inoculate.In another kind of embodiment, before the method according to this invention treatment, with prostate specific antigen (PSA) in vivo or external pre-stimulation be used for the donor white corpuscle of prostate cancer therapy.Such inoculation can be used to increase the T of predecessor cell colony and/or the T cell colony special to other tumour specific antigen, virus antigen and other pathogen antigen.

Can be used for patient for non-responsiveness with the white corpuscle of the donor of suitable antigen inoculation provides prevention or treatment to the immune defense of opportunistic infection, and these opportunistic infections such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), kaposi sarcoma-associate herpesvirus or adenovirus (Ad) infect.In a kind of example, can separate its white corpuscle to CMV antigen inoculation donor, and the method according to this invention processing, in DLI, to be used for treating the patient who suffers from cmv infection owing to immunosuppression.The patient of non-responsiveness comprises the patient who is in the immunosuppressive drug treatment, as organ transplantation patient (BM and other organ), HIV or human body, the cancer patients of EBV infection and the individuality with immunodeficient disease.

Also can enlarge the precursory cell set stimulated in vitro donor white corpuscle (being also referred to as stripped expansion).For example, in the multiple myeloma situation, before treatment blocking-up GVHD activity, by with the special MM antigen of the patient who presents by dendritic cell in external cultivation, can stimulate the donor white corpuscle.

Also can utilize stripped or stimulated in vitro expansion leukemia T cell colony to be used for the treatment of CML.In CML, (t (9 in the classical transposition of the breakpoint cluster region (bcr) of the c-abl oncogene on the karyomit(e) 9 on chromosome 22; 22) (q34; Q11)) expression of generation bcr-abl fusion gene and bcr-abl210kD fusion oncoprotein matter.The bcr-abl fused protein is that CML is special, the proteinic two kinds of antigens that main variant is a well-characterized of bcr-abl.Can carry out stimulated in vitro and expand the special T cell of bcr-abl protein, for example, as Choudhury, people such as A., blood 89:1133-1142,1997; Ten Bosch, people such as G., blood 88:3522-3527,1996 and people such as Mannering, blood 90:290-297,1997 is described.The Toplink of representing breakpoint district or contact (new contact aminoacid sequence) is as immunogen, be used for to the human T-cell who derives from healthy donors external immunity (referring to, ten Bosch, people such as G., 1996, as mentioned; People such as Mannering, 1997, as mentioned).The CD4 that produces ⁺The T cell recognition derives from allogeneic CML patient's bcr-abl express cell.In another approach, can be from CML peripheral blood of patients cell external generation dendritic cell (DC), and as antigen presenting cell be used for leukemia T cell stripped expansion (people such as Choudhury, 1997, as mentioned).In general, antigen of donor is the antigen pulse that delivery cell (APC) can be used performance disease cell characteristic, perhaps can cultivate with disease cell (for example, the tumour cell of deactivation) is collaborative; Can before input donor white corpuscle in acceptor, make these APC contact donor white corpuscles then.In addition, the direct T cell colony that can be used for expanding in the donor lymphocyte that contacts of donor white corpuscle and disease cell (or disease cell antigen).

" antigen " that is used for expanding the T cell can be polypeptide, lipid or carbohydrate part (or any combination, as glycoprotein), and can separation or reorganization produce from disease cell or pathogenic agent.The antigen that contains polypeptide does not need to form whole length protein, as long as it comprises at least a epi-position.This antigen can be contained in the vaccine composition as full length protein or its fragment or the fused protein that produces as reorganization, can with or do not combine or otherwise present with a kind of adjuvant.Vaccine can adopt various ways: the disease cell of antigen expressed, or its cell membrane preparation; Perhaps pathogenic agent is preferably the deactivation form.The method for preparing vaccine has instruction in the literature, referring to, for example, Harlow, as mentioned.Immune and external (exsomatizing) stimulates and the method for precursory cell colony expansion is known in the art, referring to, for example, Choudhury, people such as A., blood 89:1133-1142,1997; Ten Bosch, people such as G., blood 88:3522-3527,1996; People such as Mannering, blood 90:290-297,1997.

For using in the body, white corpuscle is generally used in blood plasma, synthetic medium or other physiological buffer solution, and dosage is the every kg of each infusion about 10 ⁵-10 ¹¹Individual white corpuscle, volume are approximately 50-500ml or more.These parameters, and are decided by treatment patient's doctor with disease and different with treatment.Standard DLI practice is with every kg body weight 10 ⁷-10 ⁸The dosage of individual cell is used white corpuscle.Can combine with chemotherapy and implement DLI.

For using in the body, white corpuscle of the present invention generally is that intravenously is used.

GVHD is normally obvious in 90 days behind BMT.In order to alleviate the disease among the cancer return patient, preferably after relapse diagnosis, carry out DLI in 1 week-March.For the adoptive immunotherapy behind the allosome BMT, can be from after several years, carrying out DLI before the BMT as keeping treatment.For accepting the cancer patients that DLI replaces BMT, preferably promptly impose DLI in the near future at cancer diagnosis.When not carrying out BMT, can before chemistry and radiation scheme or other treatment plan, during or carry out DLI afterwards.In all indexs, the appropriate time arrangement of DLI will be by determining doctor skilled aspect the disease treatment.

Treat successful evaluation: behind the DLI, with regard to monitoring host of the GVHD after the ordinary method or patient.GVHD is normally tangible in 90 days behind infusion.Clinical GVHD symptom comprises fash, swelling and liver, intestines, lung and joint injury, serious diarrhoea and jaundice.The level of patient's bilirubin and liver function enzyme can be measured, and also the examination of living tissue of liver can be carried out.The detailed medical history of patient-monitoring, physical examination, the complex laboratory evaluation to accepting DLI regularly comprises complete cytometry and difference cytometry, urinalysis, blood urea nitrogen creatinine, bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase, Na ⁺, K ⁺, Cl ^-, white protein, gross protein, glucose and radiography inspection.Cancer condition can be estimated by for example marrow extraction inspection and examination of living tissue, cytogenetics inspection (comprising immunohistochemical analysis) and analysis of molecules.

For the present invention, if the improvement of the symptom that visible or measurable disease symptoms or disease cause is arranged, then the treated leukocytic acceptor state of an illness of the present invention is alleviated (state of an illness refers to malignant tumour or infection).Symptom and the method for estimating its improvement are different with the state of an illness, but are familiar with by the clinician.Be used for estimating a kind of useful terminal point for the treatment of successfully and be 100 days mortality ratio.

Method and composition of the present invention also can be used for the prevention of GVHD in the non-DLI situation.These include but not limited to: thrombocyte transfer and the platelet storage process in cytokine the accumulation heating, the nonhemolytic transfusion reaction that cause.

Present method and white corpuscle treated, that do not breed of the present invention also have external purposes.For example, the PCT method is at primary culture or has that to suppress DNA in the clone (as progenitor cell and stem cell) of differentiation capability synthetic and do not influence the minimum invasive method of cell biological synthetic.Therefore, can prepare isolating stem cell, white corpuscle or other clone with treatment condition in test, thereby detect cytodifferentiation or grow step whether depend on that propagation/DNA is synthetic.These researchs can be identified along the target molecule of differentiation or development pathway, and help suppressing or promoting the development of the medicine of differentiation.For example, can stimulate the immunne response of the homogeneous variant cell that multiplication capacity is arranged, and irritation cell itself not bred simultaneously with treated white corpuscle as the irritation cell in the unidirectional MLR test yet.Use can form the PCT and the relevant processing of the compound of covalent linkage with DNA, with the application that replaces when previous irradiation or ametycin, realizes that the cell of irritation cell suppresses (cytostatis).For external application, can provide the treated white corpuscle or the composition of other cell separately or as a part of measuring test kit.This mensuration test kit can be used for MLR or be used for differentiation assays.For this purpose, treated cell generally provides with frozen form.In addition, test kit can be provided for preparing white corpuscle or have the reagent and the specification sheets of other cell of above-mentioned feature.These reagent comprise that one or more can form the compound of covalent linkage with DNA.For example, but the test kit of a kind of PCT of being used for will comprise one or more light-activated compounds, be preferably PAP or acridine, more preferably be S-59.

The following example is for the present invention described herein being described rather than limiting its scope.Some modification to this method is easy to for a person skilled in the art understand, and is contained in claims.

The multiple experimental evidence of embodiment ³The proliferation assay that the H thymidine mixes

In a kind of typical case measures, in the existence of test compounds (for example dna replication dna inhibitor or mitogen) or not, for some time of in 96 orifice plates, cell cultures being expected.Use about 20Ci/mmol ratio to live ³The preparation of H thymidine generally contains the medium in 1 μ Ci/50 μ l/ hole.This medium/marker is added in the cell, and with cell in 37 ℃ of about 4-6 of incubation hours or longer.For simplicity, with a kind of hyperchannel cell harvestor with cell transfer to filter paper, and wash free (uncorporated) marker off.Dry then this filter disc is transferred to bottle, and is dipped in the scintillation solution.Bottle can be placed auto-counter then, count the tritium gamut, determine the per minute average counter (cpm) of triplicate sample.The mensuration of compound-dna adduct

With ³The PAP of H mark measures the degree of dna modification.In the white corpuscle sample of purifying, add PAP to the final concentration of wishing.With suitable UV line dose irradiation small-sized-20mL equal portions in the PL2410 plastic containers.Control sample is only handled with PAP when no UVA.After the irradiation, purifying white corpuscle DNA.The dna content of each sample of absorbance measurement by measuring 260nm.Calculate the quantity of the psoralene adducts of per 1000 base pairs (bp) by the radioactivity of DNA sample.Typical curve makes ³The amount of H counting is relevant with the quantity of adducts.The PCR inhibition test

Polymerase chain reaction (PCR) method is well known in the art.Referring to people such as K.B.Mullis, U.S. Patent number 4,683,195 and 4,683,202, be hereby incorporated by.Available this tested and estimated the effect of PAP-DNA adducts to the template function of specific nucleic acid sequence.

For obtain in the HLA-DQ α locus the 242bp sequence or to the 439bp sequence in the beta-globin locus, the DNA sample (1 μ g) that pcr amplification is obtained by platelet concentrate photochemical treatment or gamma-radiation.Serial dilution (1: 10) is increased then and is contrasted (untreated) DNA.The DNA that handles of amplification dilutedly.Pcr amplification carries out 35 circulations.This test provides a kind of method, and the functional T cell inhibitory effect that is used for cell proliferation test is measured is associated with the direct modification of nucleic acid.

The following example 1-4 shows, PCT is relevant to the inhibition of the modification of the influence of processed white corpuscle character and white corpuscle DNA and polymerase activity.Secondly, leukocytic PCT in the platelet concentrate is handled to prevent that mouse from transferring and transfer activated GVHD in the model.

Embodiment 1

The photochemistry deactivation of T cell

Embodiment 1 proof, PCT can be with the mode deactivation white corpuscle of dependent dose.As an example, psoralene can use S-59, AMT and 8-MOP.The dose-dependently of PCT is different with the character of used psoralene, S-59 be try the most effective.

Application S-59 (square), 4 '-aminomethyl 4 have been characterized, 5 ', the photochemical treatment of 8-trimethylpsoralen [AMT] (trilateral) and 8-methoxypsoralen [8-MOP] (circle) the results are shown among Fig. 1 the relevant effect of leukocytic dosage in the platelet concentrate (PC).Platelet concentrate such as U.S. Patent number 5,593,823 described preparation and processing, this patent is hereby incorporated by.In LDA test, will come from photochemical treatment and untreated merging at random the white corpuscle of donor PC be added on the plate.At erg-ten/cm ²The constant light dosage of UVA uses three kinds of psoralenes of different concns down.With 0.05 μ M S-59,1.0 μ M AMT and 10.0 μ M 8-MOP deactivation T cells detection lower limit (marking) with arrow to LDA.These data clearly illustrate that, aspect suppressor T cell propagation, compare with 8-MOP with AMT, and S-59 is better light reagent.

Embodiment 2

Mixed leucocyte reaction

From 4 individualities, extract peripheral blood and place antithrombotics acid citrate dextrose (ACD) test tube.By the density gradient centrifugation separating periphery blood monocytic cell (PBMC) on Ficoll.The PBMC that merges three donors is as the allosome stimulator.The individual PBMC of residue is used as effector.Stimulator and effector PBMC wash twice with the RPMI that adds 10% foetal calf serum (FBS), 2mM L-glutaminate, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates, are resuspended in the supplemented medium.

Make stimulator PBMC be exposed to blood bank ¹³⁷Under the 2500cGy gamma-radiation of caesium radiator.Then with the stimulator cell to contain 1.0 * 10 ⁵100 μ L equal portions of individual cell are added in the 96 hole circle base plates.

The following PCT that stands to use S-59 of effector cell.The effector cell is resuspended in the above-mentioned cell culture medium of 30mL, and in cell suspension, adds water-based S-59 stock solution (being dissolved in the water) to Fig. 2, Fig. 3 or final concentration illustrated in fig. 4.The concentration of S-59 stock solution is different according to the S-59 final concentration of wishing with the use volume.The 30mL cell solution that will contain S-59 is then transferred in the little PL2410 bag, uses 3J/cm ²The UVA line irradiation of dosage.For Fig. 3, light dosage is 3.0J/cm ²Eccentric cell is resuspended in the cell culture medium then, to contain 1.0 * 10 ⁵100 μ L equal portions splices of individual cell, and with the stimulator cytomixis be the final volume of every hole 200 μ L.

Then at 37 ℃, 5%CO ₂Following incubation culture plate.Incubation takes out the supernatant samples of MLR after 1,2 and 7 days, be stored in-80 ℃, is used for cytokine analysis subsequently.Be used for measuring the hole incubation 6 days of cell proliferation.Behind 6 days incubations, be used for measuring the porose acceptance 1 μ Ci of cell proliferation ³The H thymidine. ³The H thymidine exists down incubation after 24 hours, with cell harvesting on glass fiber paper.Quantitatively mix by liquid scintillation counting(LSC) then ³The amount of H thymidine.Fig. 2 has shown with the effector cell's of different concns S-59 photochemical treatment ³The result that the H thymidine mixes.

Use R﹠amp; The ELISA that D Systems provides measures test kit and carries out the cytokine IL-2 and the analysis of IFN-γ synthetic of processed cell according to working instructions.Fig. 3 and Fig. 4 show the result that IL-2 and interferon-produce respectively.Among Fig. 4, the cell culture medium of taking in the 7th day is carried out IFN-γ analyze.

Embodiment 3

The leukocyte function of PCT is regulated

Embodiment 3 shows that the psoralene concentration and the light dosage that are used for PCT can be regulated synthetic and surface markers expression (for example CD69 lymphocyte activation marker expression) definite white corpuscle activity according to cytokine.These data provide evidence to show, but the concentration of energy titration UVA light dosage and light-activated compounds, obtain for the T cell proliferation in the blocking leukocyte colony and keep all effective proper ranges of white corpuscle activity, synthetic and surface markers is expressed and is proved as cytokine.A.PCT pair cell factor synthetic is regulated

With 0.5J/cm ²The constant light dosage of UVA has also been measured the synthetic (see figure 5) of IL-8 after with the PCT of different concns S-59.It is synthetic that discovery uses the PCT that increases gradually with S-59 concentration can regulate IL-8.In the S-59 concentration range with 1.0J/cm ²Also obtain similar behavior (data not shown) during UVA.These results show that the synthetic psoralene concentration and the light dosage that can be used to PCT of cytokine regulated.B.PCT handles the adjusting that CD69 expresses on the white corpuscle of back

By means of fluorescence antibody and the FACS scanning analysis of CD69, measure by PMA and Calcium ionophore and activate the induce (see figure 6) of back white corpuscle the CD69 expression.With different concns AMT and 1J/cm ²Measure the expression of CD69 after the UVA photochemical treatment over time.Find that also PCT is to rely on psoralene dosage to the influence of CD69 induced expression, when AMT concentration was more and more higher, the expression of CD69 showed bigger inhibition.When in this research, being the low dosage of S-59, induce to remain unchanged.

Embodiment 4

The dna modification that PCT causes

Embodiment 4 shows that PCT is relevant with the inhibition of white corpuscle dna modification and polymerase activity to the influence of processing white corpuscle character.

Utilize ³The radiolabeled S-59 of H, AMT and 8-MOP add 1.9J/cm ²UVA and to the scintillation counting of treated leukocytic DNA isolation, at the psoralene adducts of measuring after the photochemical treatment of the PC of donor at random that merges on the white corpuscle genomic dna (Fig. 7).Photochemical treatment with 150 μ M S-59, AMT and 8-MOP is induced 12.0,6.0 and 0.7 adductss/1000bp DNA respectively.Behind the PCT on the processed white corpuscle genomic dna relative populations of adducts relevant with PCT to the influence of leukocyte function, and prove that this influence is the direct result of dna modification.In the PCR inhibition test inhibition (data not shown) of archaeal dna polymerase DNA cloning is shown identical dependency to used psoralene concentration in the PCT process.

The result who is drawn by all biology that are used herein to the influence of research PCT dialogue cells in vitro and molecular parameter is consistent.To the estimation of leukocyte function, the mensuration of cytokine generation and the quantitative demonstration of psoralene inductive dna damage, PCT is with dose-dependent mode deactivation white corpuscle by limiting dilution analysis.Use suitable dosage, can control the expression of leukocytic function such as production of cytokines or antigenicity mark.In addition, in three kinds of psoralenes that tried, S-59 is being the most effective aspect the deactivation white corpuscle.The dosage range of S-59 suppressor T cell propagation is approximately 3.5log unit.Therefore, can suppress white corpuscle propagation and the GVHD that follows but the dosage window that keeps immunologic function greater than other ablation method provided.

Embodiment 5

Mouse is transferred the prevention of TA-GVHD in the model

Following mouse is transferred the model that model is the anthropomorphic dummy TA-GVHD clinical syndrome of fine sign (Fast, people such as LD, blood 82:292,1993).According to the in vitro results of expection, use this body inner model, by parent (A strain, the H-2 of will isozygotying ^a) splenocyte transfer immunocompetence heterozygosis F ₁Hybrid receptor (B6AF1 strain, H-2 ^A/b) in, estimate the influence that the S-59 photochemical treatment suppresses TA-GVHD.

Through outside tail vein with about 10 ⁸Individual donor (A or F ₁) splenocyte imports each acceptor (F ₁) in.Be used as the source of the T cell of living by the splenocyte of density gradient centrifugation acquisition.Use three and transfer group: (1) handles control group (F ₁→ F ₁): with B6AF ₁Splenocyte input B6AF ₁In the acceptor; (2) positive TA-GVHD control group (A → F ₁): with donor A splenocyte input B6AF ₁In the acceptor; (3) S-59 photochemical treatment group (PCT A → F ₁): with 150 μ M S-59 and 3 joules/cm ²UVA handles the splenocyte of donor A, imports B6AF then ₁In the acceptor.

Transfer the biological evidences of back TA-GVHD of monitoring acceptor in 2 weeks.Activate the donor T Transplanted cells of flow cytometry analysis acceptor splenocyte with Two Colour Fluorescence.In this test, also use anti-H-2 with pan-T cell, anti-CD 3 antibodies ^bThe antibody staining splenic t-cell.According to anti-H-2 ^bThe shortage of antibody response detects the T cell of donor A.In control experiment, F ₁→ F ₁The positive splenocyte of CD3 above 99% of acceptor is all used anti-H-2 ^bAntibody labeling.Abreast, use ⁵¹The Cr breaking test is measured the existence of cytotoxic lymphocyte (CTL) in the acceptor spleen.With splenocyte with ⁵¹EL-4 cell (the H-2 of Cr mark ^b) with 150: 1 effector: target ratio was at 37 ℃ of following incubation 4-6 hours.According to the cracking of target cell and ⁵¹The cytotoxicity of CTL is measured in the release of Cr in substratum.

The result shows, although contrast F during this research ₁→ F ₁Acceptor in the group is still healthy, but A → F ₁Acceptor in the group develops the biology symptom of TA-GVHD after week at donor spleen cells input 2-3.TA-GVHD is characterised in that splenomegaly, donor T cell (H-2 ^a) immigration (28.6 ± 11.3%) and the acceptor spleen in CTL have (35 ± 18.7% ⁵¹The Cr cracking) (table 1).

Transfer two all backs by measuring spleen: body weight ratio is estimated splenomegaly.Transfer three all backs and detect the development of immune deficiency by the evaluation of thymocyte structure.Thymic aplasia is the reliability index of the acquired immunodeficiency relevant with TA-GVHD.

Also monitor the GVHD clinical symptom of acceptor, comprise body weight, posture, activity, skin complete, fur quality, white blood cell count(WBC), red blood cell count(RBC) and platelet count.F ₁→ F ₁The body weight weekly of healthy animal increases in time in the group, and A → F ₁The mean body weight of animal does not increase in time in the group.

A → F ₁The average clinical score of animal in the group (0-2 level, healthy animal are 0 grade) increases in time, shows on the TA-GVHD surface it is significantly also little by little to increase the weight of.PCT A → F ₁Animal in the group is similar to contrast F ₁→ F ₁Animal in the group, still healthy and do not have a visible TA-GVHD clinical manifestation.When transfer the back when surpassing for 10 weeks at A → F ₁Observe 25% mortality ratio in the group, and contrast F ₁→ F ₁With PCT A → F ₁All animals in the group are all kept fit.

Table 1

The B6AF that transfers 1The biological assessment and the cell counting of acceptor (N=5-11)
				Parameter	????F 1→F 1(mean value ± S.D.) 1	????A→F 1(mean value ± S.D.)	????PCT?A→F 1(mean value ± S.D.)
The T cell of % donor A 2	????1.1±0.7	????28.6±11.3	????1.0±0.7
				The % cracking ( 51Cr) 2	????11.5±3.3	????35±18.7	????18.0±4.2
Spleen weight/body weight (* 10 3) 2	????3.6±0.4	????25.9±1.8	????2.7±0.4
				Thymocyte counting (* 10 6) 3	????27.0±11.0	????7.5±2.9	????27±4.0
WBC counting (10 3/μl) 3	????7.6±2.8	????2.5±0.5	????7.1±1.5
				RBC counting (10 6/μl) 3	????8.8±0.3	????7.4±0.8	????8.6±0.2
Thrombocyte (10 3/μl) 2	????1248±201	????1038±167	????1291±159

1.S.D.=standard deviation 2. is transferred two all backs mensuration 3. and is transferred three all backs mensuration

Prepare tissue slice and check that its histology is unusual at blind editorial afterword.Existence and the bile duct of liver being estimated lymphatic infiltration liquid destroy and blood vessel endothelium inflammation and infiltration.With regard to the reservation of lymph folliculus or the histology of destruction evaluation spleen.The rim surface zone of evaluating skin section and the lymphatic infiltration in the adnexa.Estimate marrow each main pedigree of cutting into slices: spinal cord, cell born of the same parents class and Megakaryocytic cellularstructure and maturation.In blind test, A → F ₁Mouse is developed the histology evidence of GVHD in liver, spleen, marrow and oral mucosa.On the contrary, donorcells of photochemical treatment (PCT-A) or homologous cell (F ₁) acceptor display organization learn unusual.

With concentration is S-59 and the 3J/cm of 50nM-150 μ M ²Transferring of several groups of mouse of the input cell that UVA handles shows that in this scope GVHD suppresses completely.This dosage window class that shows that GVHD can be suppressed in the PCT body is similar to that in vitro tests obtains.

Conclusions external and the interior data of body are in the S-59 of 3.5 log units concentration, can suppress its propagation and suppress TA-GVHD in vivo external leukocytic PCT processing.For identical concentration range, observe the adjusting of the dependent dose of external leukocyte function (the synthetic and surface molecular expression of cytokine).

Embodiment 6

Present embodiment has been described a kind of method, is used for determining to be suitable for suppressing GVHD and the PCT condition that keeps the GVL effect of DLI, for example is used for the treatment of recurrence leukaemic after to allogeneic BMT.Experiment purpose is summarized as follows.

A. determining of the psoralene concentration range of using in PCT preventing external lymphocytic propagation, thereby suppresses GVHD.

B. the sign of the white corpuscle phenotype of external photochemical treatment produces the T cell of GVL effect in the body and the expression of NK cell-surface antigens to determine cell survival and indication.

C. to the qualitative and quantitative analysis of external treated leukocytic cytokine-expressing.

D. the proof that treated white corpuscle-DLI suppresses GVHD in the body in the murine leukemia model.With psoralene to leukocytic photochemical treatment

The stock solution of preparation psoralene as described below.AMT stock solution by dissolving 50mgAMT powdered preparation 15mM in 10mL distilled water.Violent this solution that mixes, and by 0.2 μ m syringe-type membrane filtration.Use the absorbancy of Shimadzu UV160U spectrophotometric determination solution, determine the concentration of AMT in the filtered solution at 250nm.Use 25000M ^-1Cm ^-1The optical extinction coefficient value calculate the concentration of AMT.

Stock solution by dissolving S-59 powdered preparation S-59 psoralene in distilled water.Violent this solution that mixes, and by 0.2 μ m syringe-type membrane filtration.Use the absorbancy of Shimadzu UV160U spectrophotometric determination solution, measure the concentration of S-59 in the filtered soln at 250nm.Use 25400M ^-1Cm ^-1The optical extinction coefficient value calculate the concentration of S-59.

8-methoxypsoralen (8-MOP) is that solubleness is relatively poor in the aqueous solution.Therefore, use is with the saturated PAS solution (PAS of 8-MOP, can obtain from Baxter, be a kind of artificial blood surrogate, is made up of sodium acetate, trisodium citrate, the sodium-chlor phosphoric acid buffer of pH7.2,300mOsm) the preparation white corpuscle preparation of 8-MOP and UV treatment.Prepare its saturated solution by suspension 100mg 8-MOP in 100mLPAS.In dark container under room temperature with this solution stirring 16 hours.The solution that obtains by 0.2 μ m membrane filtration is to remove any insoluble 8-MOP.Use the absorbancy of Shimadzu UV160U spectrophotometric determination 248nm, measure the final concentration of 8-MOP.Use 22900M ^-1Cm ^-1The optical extinction coefficient value calculate the concentration of 8-MOP.

In the donor leukocyte population suspension of purifying, add the psoralene stock solution to the final concentration of wishing.By above-mentioned preparation method, the unit that uses the saturated PAS preparation of 8-MOP to handle with 8-MOP.In the photoactivation device, shine the donor white corpuscle to 10 of purifying with the UVA line ^-3-100J/cm ²Whole dosage.In irradiation process, stir this unit with 70 rev/mins.Before irradiation, take the white corpuscle sample with comparing.

With 10 ^-4The concentration of-150 μ M adds S-59 in the white corpuscle preparation of purifying.White corpuscle will have 10-10 ⁸Cell/mL, preferably 10 ⁷Cell/mL, more preferably 2 * 10 ⁶The cell density of cell/mL.

Make that to be exposed to wavelength with S-59 blended leukocyte population sample be 200-450nm, preferably under the ultraviolet ray of 320-400nm.Cell preferably accepts about 10 ^-3-100J/cm ²Light dosage.In a kind of preferred embodiment, light dosage is 3J/cm ²The time limit that the UV line exposes is about 1 second to 60 minutes, preferably about 1 minute.

Behind the PCT, as above reach described in the embodiment, measure white corpuscle sample propagation and participate in the active ability of white corpuscle.Should measure following parameters: cell survival and integrity; Proliferation activity; Surface antigen is expressed; Cytokine secretion; GVHD and GVL activity.

Following test can be used for alkylating agent white corpuscle that handle or photochemical treatment.A. limiting dilution test (LDA) external leukocytic dose response kinetics i)

This tests the T cell quantity that direct mensuration can be cloned, its sickness rate relevant with severity (Keman, people such as N.A., blood 68:770-773,1986) direct in vivo and GVHD.FDA uses LDA that the gamma-radiation of blood products is set up general purpose discipline.This is tested according to Pelsynski, people such as M., and blood 83:1683-1689,1994 method is carried out.In brief, handling the quantity that the human leukocyte proliferative cell is measured in the back with different concns medicine and constant dosage light PCT.Allosome irritation cell with the lethality gamma-irradiation that merges comes stimulating growth.The propagation contrast comprises untreated white corpuscle (as the positive control of propagation) and accepts the preceding white corpuscle (negative control) of PCT of lethality gamma-irradiation.Quantitative measurement growth according to isolating propagation colony.To human leukocyte measure used dosage and the cell count that suppressed by propagation between dependency.The dose response that obtains from this experiment estimate to be similar to for the platelet concentrate white corpuscle obtained.

The following LDA that carries out.From peripheral blood, separate white corpuscle by the white corpuscle electrophoresis.The thorn activated T cell makes it to breed in the hole of the microtiter plate that contains the RPMI substratum, has added foetal calf serum (FBS), phytohemagglutinin (PHA), reconstituted inter leukin-2 (rIL-2) and T cell growth factor (TCGF) in the substratum.Also contain in each hole from the PBMC of 10 individualities and merge the thing preparation and with 10 of 5000cGy gammairradiation ⁵Individual allosome irritation cell.

With the white corpuscle of each untreated control sample with two serial dilutions independently.In every hole, add and contain 10 of a series ⁵, 10 ⁴, 10 ³, 10 ², 10 ¹With 10 ⁰100 μ L equal portions of 300,100,33,11 and 4 cells of individual cell or second series.Each extent of dilution adds in the entering plate with 10 repeated sample.Cultivation contains 1.1 * 10 ⁷The 11mL equal portions of the sample of the photochemical treatment of individual total leukocyte detect T cell alive.100 μ L equal portions of this sample are not added in 110 holes dilutedly.

With microtiter plate at CO ₂37 ℃ of 3 weeks of incubation in the incubator.Incubation 0.5,1 and after 2 weeks is raised with TCGF, FBS, rIL-2 and PHA every hole.Last in 3 all incubation period marked to every hole with regard to the existence of T cell clone.The hole scoring that will contain one or more T cell clones is positive.The hole that does not contain the T cell clone is marked negative.Use T cell frequency based on each sample of minimum X analytical calculation of Poisson's distribution.Calculate the T cell with following formula and reduce coefficient: the T cell reduces coefficient=f _Contrast/ f _Handle, f wherein _ContrastAnd f _HandleBe respectively the T cell frequency of contrast and treated sample.

Contrast equal portions or do not handle or only with UVA or only handle with S-59.Equal portions are with the gammairradiation of 2500cGy clinical dosage.In all the other equal portions, add S-59 to final concentration be 10 ^-4μ M-150 μ M.Then with 10 ^-3-100 joules/cm ²UV light dosage irradiation.Ii) mixed lymphocyte reacion (MLR) test

This test is also referred to as MLC test, according to Kraemer, and people such as K.H., dermatological studies magazine 77:235-239,1981 and Kraemer, people such as K.H., dermatological studies magazine 76:80-87,1981 method is carried out.As for the ultimate principle and the control of this test, referring to " basis and clinical immunology ", the 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow (volume), Appleton ﹠amp; Lange, Norwalk, CT, 1994,246-247 page or leaf.DNA is synthetic in its indirect measurement allosome stimulation inductive lymphocyte.When merging the lymphocyte of the different individuality of two kinds of HLA-in tissue culture, cell expands, synthetic DNA and propagation, and the identical cell of HLA-is still static.According to ³H-thymidine mensuration inductive DNA that mixes of nucleic acid in proliferative cell synthesizes.Harvested cell then, the unconjugated radioactivity of flush away, and in the β counter radioactivity of counting internalization.

In single channel MLR, measure leukocytic effector and stimulator function.Comprise standard, the suitable positive and negative control.By the effector cell is measured effector function with merging human leukocyte (irritation cell) incubation of lethality gamma-irradiation.After the lethality gamma-irradiation is with the picked-up of blocking-up thymidine, by measure the stimulatory function of processed white corpuscle (with PCT or other alkylating agent compound treatment) with the human leukocyte incubation of normal merging.The result who becomes with processing dosage who obtains is compared with two pass MLR.

Although MLR will provide the part of the information that LDA provides, it can provide the information about the ability of processed other cell of leukocyte stimulation.Processed leukocytic stimulation can be as a kind of model of leukemia immune response inducing among the MLR, and it transfers among the patient at BMT and is activated by DLI.In this situation, host that antileukemie immunne response (GVL effect) can be existed among the BMT or donorcells starting.B. be subject to processing leukocytic phenotypic characteristic i) the surface antigen marker expression

Utilize the influence of suitable fluorescent-labeled antibody (Pharmingen) and standard FACS-SCAN assay determination PCT or alkylating agent processing to surperficial antigenicity marker expression.As Coligan, people such as J.E., " immunology general scheme: general scheme ", New York, John Wiley ﹠amp; Sons, Inc., described in 1991, utilize suitable fluorescent-labeled antibody (Pharmingen) and standard FACS-SCAN to analyze, as the existence of function mensuration antigenicity mark CD2, CD28, CTLA4, CD40 part (gp39), CD18, CD25, CD69 and the CD16/CD56 of treatment dosage, known these marks participate in and T cell and NK cell-stimulating and the relevant interactions of immunologic function.If it is, then unaffected at the stability prediction of treatment condition lower surface molecule to proteinic PCT and relevant treatment character gentleness; Yet inducing of its expression estimates it is dose-dependent.

About processed human lymphocyte express in propagation, surface markers, cytokine is synthetic and cytotoxicity aspect sign, see embodiment 14.Ii) the leukemia cell is cracking

In order to measure the direct GVL effect that is subject to processing cell induction, research is to the molten cell ability of a series of people and L-1210 (can obtain from ATCC).According to Jiang, people such as Y.Z., bone marrow transplantation 8:233-238,1991 condition is carried out this experiment.As Coligan, people such as J.E., " immunology general scheme: general scheme ", New York, John Wiley ﹠amp; Sons, Inc., 1991 is described, uses under standard conditions ⁵¹Cr mark leukemia cell.In supernatant liquor ⁵¹All cracking that reach are measured in the radioactive release of Cr, and with as a setting and total cracked contrast compare.The monitoring treatment dosage is to the influence of the molten cell ability that is subject to processing the T cell.Treated white corpuscle by adding cumulative quantity is also observed and whether cracking takes place is carried out this experiment.Can repeat this experiment with the T cell subsets.Although the direct cracking of leukemia system provides a kind of direct mechanism of GVL effect, its shortage may mean that DLI induces a kind of indirect mechanism of GVL effect.Therefore, in addition, can measure cytotoxicity as the generation of IFN-γ, IL-2 or GM-CSF by measuring the cytokine generation.Iii) cells in vivo viability

For the viability of cell treated in the estimated body, after the processing of various dose, the white corpuscle of male mice is imported in female homology or the heteroreceptor.The PCR method that blood sample carried out that the time point that utilization is wished after splenocyte is transferred is taked (Goodarzi, people such as M.O., blood transfusion 35:145-149,1995), the existence of treated cell in the monitoring circulation.This method detects the existence that only is present in the Y-chromosome specific gene sequence in male (donor) cell, has the susceptibility of 1-5 cell of per 50 μ L host blood.This experiment will be measured the influence to the removing of input cell in the Live Animals, and the relation between treatment dosage and the cells in vivo circulation.As people such as Johnson, Scandinavia Journal of Immunology (Scand.J.Immunol.) 45:511-514,1997 is described, by before infusion to leukocytic PKH-26 mark also can detection bodies in treated leukocytic viability.C. the cytokine synthetic quantitatively reaches qualitative sign

Utilization can the commercial Elisa test kit (R﹠amp that obtains; D Systems), after effector cell and the collaborative cultivation of irritation cell, culture supernatant is quantitatively reached qualitative cytokine assay.Test according to the specification sheets that Elisa manufacturer provides.The typical curve that will produce the cytokine standard that the absorbance measurement of each sample and each are measured test kit and provided is compared estimated result.As the function of compound concentration and-under the situation of PCT-light dosage and handle with measure between the function in the timed interval white corpuscle of processing is carried out cytokine assay.Owing to IL-1, IL-2, IL-4, IL-10, the IFN-gamma cells factor are measured in the clearly effect in t cell activation and GVHD/GVL.

Pending white corpuscle cytokine synthetic is determined that it is important producing in the ability that works as the immune response inducing thing at the mensuration cell by cytokine; It also can be used as the mensuration to biochemical function and protein synthesis.

About processed human lymphocyte express in propagation, surface markers, cytokine is synthetic and cytotoxicity aspect sign, see embodiment 14.D. mouse model suppresses and the application of GVL effect inductive for proof GVHD in the body

For research graft versus host disease and graft leukemia effect, mouse is very perfect model.In order to estimate of the influence of treated white corpuscle respectively, use at Johnson people such as B.D., bone marrow transplantation 11:329-336,1993 and Johnson, people such as B.D., blood 85:3302-3312, the transplanting mouse model described in 1995 to GVHD and GVL.In this model, inferior (9Gy) female AKR/J mouse (H-2 that shines deadlyly ^k), give MHC-then and mate female B10.BR donor (H-2 ^k) 10 ⁷Individual BM cell adds 3 * 10 ⁷Individual splenocyte.This processes and displays causes the Acute GVHD clinical symptom and is dead in 50 days.The elimination of the splenocyte of input causes the survival fully of mixochimaera, and BMT does not cause 50% mortality ratio.In the present embodiment, before transferring, mice spleen cell is handled, detected the inhibition of handling GVHD to measure, and the lowest dose level that suppresses the GVHD morbidity also is provided according to mortality ratio.Splenocyte is the leukocytic generally acknowledged substitute of mouse blood, because the volumetric blood of mouse is for these experiments and Yan Taixiao.

For the GVL reactivity of evaluation process cell, BMT imported for example PA allosome splenocyte to acceptor after 21 days, attacked with AKR leukemia cell after 7 days then.According to known to, BMT recovers (28 days) 5 * 10, back ⁴AKR leukemia cell's mortality ratio (Johnson, people such as B.D. transplant 54:104-112,1992) of 95% after the input leukemogenesis of mouse and leukemia are attacked 25 days.On the contrary, the input of BMT allosome splenocyte after 21 days shows, can induce the GVL effect after with the attack of AKR cell after 7 days.This mechanism causes after 70 days 70% survival rate.If proof has the prevention of GVHD, the processing of splenocyte makes and can detect inducing of GVL when no GVHD before then transferring.Its mensuration is that this receptor is attacked with the AKR cell according to the survival rate of the BMT acceptor of the treated splenocyte of input.

Function as treatment dosage detects the influence of the pending leukocytic DLI of use to leukemia attack survival rate, and compares with the control group of the splenocyte infusion that is untreated.Utilize as Johnson people such as B.D., bone marrow transplantation 11:329-336,1993 described PCR assay methods, the chimerism and the leukemia load of detection survival mice.

In this experimental model, before dead, can diagnose the leukemic cause of the death, because leukemia can cause AKR leukemia cell's expansion to the white blood cell count(WBC) or the pcr analysis of blood.The primer special to AKR leukemia cell's specific marker can be used for pcr analysis.According to the death of hanging down white blood cell count(WBC) and splenatrophy and causing by distinctive clinical symptom (change of fur and posture, viscosity fecal etc.) diagnosis GVHD.By the injection still less or more substantial AKR leukemia cell detect the usefulness of PA-DLI to remaining disease of minimum or higher tumour load.

For example pass through the PCT method to leukocytic processing according to the present invention, if under the condition that does not cause GVHD, after imposing the leukemia attack, can induce and improve significantly, think that then this is effective to inducing the GVL effect with the mouse survival rate statistics of handling cell DLI processing.The genotype of surviving animals should also be fully chimeric, because this more high incidence with anosis long-term surviving is relevant in the mankind.In 100 days, the raising of survival rate is easy to proof behind initial BMT.

To handle splenocyte for donor under the leukocytic condition above-mentioned.

About the situation of the treated white corpuscle GVL effect that causes when the no GVHD in the mouse model system, see embodiment 15.

Embodiment 7-12

Following specific embodiment is for the preparation method of useful in the methods of the invention representative alkylating agent compound being described, providing about the pertinent data to the useful compound of professional, and the method for compound validity is measured in explanation.Unless propose in addition, at CDCl ₃All NMR spectrums of record on the middle Varian 200MHz instrument; Report is with respect to the chemical shift of tetramethyl silane (TMS).With Perkin Elmer FTIR record IR spectrum.With the moisture H of 5mM ₃PO ₄As the A mobile phase A, use 5mM CH ₃CN carries out HPLC with YMC C8 post with gradient mode as Mobile phase B.In DMSO or ethanol, prepare sample, and before injection, remain in≤15 ℃.

Table 2 shows the title of the compound number that is used for different compounds.

Table 2

Compound number	Chemical name
		IV	Two (2-chloroethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] ethyl ester
V	Two (2-chloroethyl) amino of N-(acridine-9-yl) Beta-alanine 2-[] ethyl ester
		VI	Two (2-chloroethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) 4-aminobutyric acid 2-[] ethyl ester
VII	Two (2-chloroethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) 5-aminovaleric acid 2-[] ethyl ester
		VIII	Two (2-chloroethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 3-[] propyl ester
IX	Two (2-chloroethyl) amino of N-(4-methoxyl group acridine-9-yl) Beta-alanine 2-[] ethyl ester
		X	Two (2-chloroethyl) amino of N-(3-chloro-4-methylacridine-9-yl) Beta-alanine 2-[] ethyl ester
XI	[N, N-two (2-chloroethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] propyl ester
		XII	[N, N-two (2-chloroethyl)] Beta-alanine 2-[(6-chloro-2-methoxyl group acridine-9-yl) amino] ethyl ester
XIII	Two (2-chloroethyl) amino of N-(6-chloro-2-methoxyl group acridine-9-yl) Beta-alanine 2-[] ethyl ester
		XIV	[N, N-two (2-chloroethyl)]-2-aminoethyl 4,5 ', 8-trimethylammonium-4 '-acetate psoralene

Two (2-chloroethyl) amino of embodiment 7N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] two (2-hydroxyethyl) amino of synthesis step A.N-(tertbutyloxycarbonyl) Beta-alanine 2-[of ethyl ester dihydrochloride (compound IV)] ethyl ester

At N ₂Down in-15 ℃, N-(tertbutyloxycarbonyl)-Beta-alanine (20.3g in doing THF, 107mmol) with 4-methylmorpholine (13.0mL, 12.0g, add chloroformic acid isobutyl (13.9mL in stirred solution 119mmol) (200mL), 14.6g, 107mmol), cause the formation immediately of white precipitate (4-methylmorpholine HCl).-15 ℃ of following stirred reaction mixtures 5 minutes, subsequently reaction mixture is transferred in the flask, contain-15 ℃ of trolamine (48.3g, stirred solutions 324mmol) (150mL) of doing down among the THF in this flask.Reaction mixture is warmed to 23 ℃, and stirred other 1.5 hours, remove precipitation by vacuum filtration subsequently.From filtrate, remove THF then in a vacuum, (distribute remaining viscosity yellow oil between 5 * 150mL) at water (500mL) and EtOAc.At Na ₂SO ₄The last dry organic layer that merges.The removal of solvent produces the hope product of 25.8g (75%) in the vacuum: two (2-hydroxyethyl) amino of N-(tertbutyloxycarbonyl) Beta-alanine 2-[] ethyl ester, be a kind of luteotestaceous oil. ¹H NMR: δ 5.32 (brs, 1H), 4.18 (t, J=5.4Hz, 2H), 3.58 (t, J=5.1Hz, 4H), 3.37-3.23 (m, 2H), 2.80 (t, J=5.4Hz, 2H), 2.69 (t, J=5.1Hz, 4H), 2.51 (2H), 1.41 (s 9H) does not find hydroxyl proton for t, J=6.0Hz. ¹³CNMR: δ 173.0,156.4,79.8,63.3,60.2,57.3,54.1,36.7,35.3, two (the 2-tert-butyl dimetylsilyl oxygen ethyl) amino of 28.8. step B.N-(tertbutyloxycarbonyl) Beta-alanine 2-[] ethyl ester

At N ₂Two (2-hydroxyethyl) amino of N-(tertbutyloxycarbonyl) the Beta-alanine 2-[that will obtain by steps A down] (22.7g, 70.9mmol) (11.1g, acetonitrile stirred solution (70mL) 163mmol) is cooled to 0 ℃ to ethyl ester with imidazoles.(534mg 3.54mmol), and stirs reaction mixture other 5 minutes under 0 ℃ to add TERT-BUTYL DIMETHYL CHLORO SILANE then.Reaction mixture is warmed to 23 ℃, stirred 2 hours, remove the white precipitate (imidazoles HCl) of generation subsequently by vacuum filtration.From filtrate, remove acetonitrile in a vacuum, (distribute remaining material between 3 * 200mL) at saturated brine (600mL) and EtOAc.At Na ₂SO ₄The last dry organic layer that merges.The removal of solvent produces the hope product of 35.2g (90%) in the vacuum: two (the 2-t-butyldimethylsilyl oxygen ethyl) amino of N-(tertbutyloxycarbonyl) Beta-alanine 2-[] ethyl ester, be a kind of xanchromatic oil. ¹HNMR: δ 5.29 (brs, 1H), 4.14 (t, J=6.0Hz, 2H), 3.65 (t, J=6.3Hz, 4H), 3.37 (apparent q, 2H), 2.85 (t, J=6.0Hz, 2H), 2.71 (t, J=6.3Hz, 4H), 2.49 (t, J=5.9Hz, 2 H), 1.42 (s, 9H), 0.88 (s, 18H), 0.03 (s, 12H); ¹³C NMR: δ 172.7,156.3,79.7,63.3,62.4,57.7,54.3,36.7,35.3,28.9,26.4,18.7, two (the 2-t-butyldimethylsilyl oxygen ethyl) amino of-4.9. step C. Beta-alanine 2-[] ethyl ester

To containing two (the 2-t-butyldimethylsilyl oxygen ethyl) amino of N-(tertbutyloxycarbonyl) Beta-alanine 2-[that obtain from step B] (3.01g in flask 5.48mmol), adds pure trifluoroacetic acid (5mL) to ethyl ester, causes CO ₂Emitting of gas.Stirred reaction mixture 5 minutes is removed trifluoroacetic acid in a vacuum.At saturated NaHCO ₃(100mL) and EtOAc (distribute remaining material between 3 * 30mL).At Na ₂SO ₄The last dry organic layer that merges.The removal of solvent produces the hope product of 2.45g (100%) in the vacuum: two (uncle 2--Ding dimethylsilane oxygen ethyl) amino of Beta-alanine 2-[] ethyl ester, be a kind of luteotestaceous oil. ¹H NMR: δ 4.12 (t, J=6.0Hz, 2H), 3.63 (t, J=6.4Hz, 4H), 2.96 (t, J=6.2Hz, 2H), 2.84 (t, J=6.0Hz, 2H), 2.69 (t, J=6.4Hz, 4H), 2.44 (2H), 0.86 (s, 18H), 0.03 (s 12H) does not find the amine proton for t, J=6.2Hz. ¹³C NMR (CDCl ₃): δ 173.0,63.4,62.6,57.9,54.4,38.4,38.1,26.4,18.7, two (2-hydroxyethyl) amino of-4.9. step D.N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] ethyl ester

By under the room temperature at 10mL CHCl ₃The middle stirring 12.5 hours makes two (the 2-t-butyldimethylsilyl oxygen ethyl) amino of Beta-alanine 2-[] (736mg is 1.64mmol) with 9-methoxyl group acridine-2-carboxylate methyl ester (669mg, 2.50mmol) reaction for ethyl ester.Filter out precipitation (dihydroketoacridine) then, at saturated water-based NaHCO ₃(100mL) and CHCl ₃(distribute filtered solution between 3 * 35mL).At Na ₂SO ₄The last dry organic layer that merges, and concentrate in a vacuum, the oil of 1.61g viscosity brown produced.By at N ₂Down thick intermediate is dissolved among the 3.0mL THF, and after being cooled to 0 ℃, uses HF/ pyridine (1.0mL) to handle immediately, carry out deprotection the glycol that produces.Stir and made solution be warming up to room temperature in 1 hour.Remove volatile matter in a vacuum, at saturated water-based NaHCO ₃(100mL) and CHCl ₃(distribute resistates between 3 * 35mL).The dry organic layer that merges concentrates and produces the filemot solid of 649mg.Preparation type TLC (C-18, CH ₃CN) obtain the glycol of the hope of 20% productive rate, two (2-hydroxyethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] ethyl ester (according to HPLC, purity＞80%); ¹H NMR: δ 8.82 (s, 1H), 8.21-7.94 (m, 2H), 7.94-7.72 (m, 2H), 7.59 (apparent t, 1H), 7.23 (apparent t, 1H), 4.30-4.18 (m, 2H), 4.18-4.05 (m, 2H), 3.89 (s, 3H), 3.69-3.50 (m, 4H), 2.92-2.73 (m, 4H), 2.73-2.55 (m, 4H) two (2-chloroethyl) amino of step e .N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride

Two (2-hydroxyethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] ethyl ester is to the conversion of dichloro compound, is that (Journal of the American Chemical Society (J.Am.Chem.Soc.) 1959, method 81:3984) realizes by being similar to people such as Peck.To be dissolved in pure SOCl ₂(41mg, yellow solution 0.090mmol) at room temperature stirred 20 hours the product that obtains from step D (6mL).Remove SOCl then in a vacuum ₂Obtain a kind of yellow solid (dihydrochloride).Then at saturated NaHCO ₃(50mL) and CH ₂Cl ₂(distribute this material between 3 * 20mL).At Na ₂SO ₄The last dry organic layer that merges.The removal of solvent produces the dichloro compound of 35.4mg free alkali in a vacuum, is a kind of orange glue. ¹H NMR: δ 8.82 (s, 1H), 8.20-7.83 (m, 4H), 7.5 (apparent t, 1H), 7.25 (apparent t, 1H), 4.36-4.15 (m, 4H), 3.93 (s, 3H), 3.48 (t, J=6.9Hz, 4H), (m, 4H), 2.86 (t, J=6.9Hz 4H) do not find the amine proton to 3.06-2.77. ¹³CNMR：δ172.3，166.6，155.2，146.5，144.6，133.1，131.6，128.7，124.6，124.3，116.1，114.3，63.7，57.2，53.5，52.9，46.3，42.5，35.2.

Do not find other carbon.By adding HCl in the 1M ether from CH ₂Cl ₂Middle precipitation HCl salt obtains two (2-chloroethyl) amino of N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 2-[] ethyl ester dihydrochloride (compound IV), be a kind of xanchromatic solid (is 81% according to HPLC purity).

Prepare two (2-chloroethyl) amino of N-(acridine-9-yl) Beta-alanine 2-[with similar methods] ethyl ester dihydrochloride (compound V).In step D, replace 9-methoxyl group acridine-2-carboxylate methyl ester, obtain intermediate glycol (7.1%) (is 74% according to HPLC purity) for a kind of yellow oil with 9-methoxyl group acridine. ¹H NMR: δ 8.14 (d, J=7.5Hz, 2H), 7.93 (d, J=8.6Hz, 2H), 7.52 (apparent t, 2H), 7.23 (apparent t, 2H), 4.36-4.08 (m, 4H), 3.76-3.5 (m, 4H), 3.08-2.60 (m, 8H)

Do not find amine and hydroxyl proton.

(37.3mg, thionyl chloride solution 0.0793mmol) (4.0mL) stirred 7.5 hours down at 23 ℃ with the intermediate glycol.Remove thionyl chloride in a vacuum, obtain a kind of xanchromatic oil.With this substance dissolves in ethanol (～4mL) in, and remove in a vacuum and desolvate.Then with this substance dissolves in CH ₂Cl ₂(4mL) and in a vacuum except that desolvating; This step repeats twice.(3 * 4mL) grind this material, and obtaining 40.0mg is the product (is 42% according to HPLC purity) of yellow water-absorbent vitreous solid form to use hexane then.By at saturated NaHCO ₃With CH ₂Cl ₂Between distribute, subsequently at Na ₂SO ₄The last dry organic layer that merges, and remove in a vacuum and desolvate, some material is converted into unhindered amina is used for analysis purposes. ¹H NMR: δ 8.21-8.00 (m, 4H), 7.66 (apparent t, 2H), 7.38 (apparent t, 2H), 4.26-4.12 (m, 2H), 4.12-3.98 (m, 2H), 3.43 (t, J=6.9Hz, 4H), 2.96-2.68 (m, 8H)

Do not find the amine proton.

As stated above, but replace N-(tertbutyloxycarbonyl)-Beta-alanine with N-(tertbutyloxycarbonyl)-4-aminobutyric acid, preparation N-[(2-methoxycarbonyl acridine-9-yl)] two (2-chloroethyl) amino of 4-aminobutyric acid 2-[] ethyl ester dihydrochloride (compound VI) (is 78% according to HPLC purity). ¹H NMR: δ 8.89 (s, 1), 8.12 (apparent t, 2), 7.93-7.80 (m, 2), 7.59 (apparent q, 1), 7.36-7.20 (m, 1), 4.16 (t, 2, J=5.7Hz), 4.07-3.92 (m, 2), (3.97 s, 3), 3.46 (t, 4, J=6.9Hz), 2.93-2.80 (m, 6), 2.60 (t, 2, J=6.5Hz), 2.29-2.12 (m, 2)

Do not find the amine proton.

Embodiment 8

Use 3-[N, N-two (2-t-butyldimethylsilyl oxygen ethyls)] aminopropanol replaces the trolamine in embodiment 7 steps A, continue two (2-chloroethyl) amino of preparation N-(2-methoxycarbonyl acridine-9-yl) Beta-alanine 3-[then from step C] propyl ester dihydrochloride (compound VIII) (is 63% according to HPLC purity). ¹H NMR: δ 8.91 (s, 1), 8.20-7.93 (m, 4), 7.18 (performance t, 1), 7.39 (apparent t, 1), 4.30 (m, 4), 3.96 (s, 3), 3.48 (t, 4, J=6.9Hz), 2.88-2.60 (m, 2), 2.83 (t, 4, J=6.9Hz), 2.62 (t, 2, J=6.7Hz), 1.85-1.68 (m, 2)

Do not find the amine proton.

Embodiment 9

The synthetic compound also can be with the preparation of following method in embodiment 7: two (2-chloroethyl) amino of N-(acridine-9-yl) Beta-alanine 2-[] ethyl ester dihydrochloride (compound V) synthetic: method II steps A .N-(acridine-9-yl) Beta-alanine methyl esters dihydrochloride

Merge 9-chloroacridine (11.7g, " organic synthesis " (Organic Synthesis), Coll. the 3rd volume, the 57th page), Beta-alanine methyl ester hydrochloride (9.9g) and sodium methylate (3.26g), add 60mL methyl alcohol.With this mixture of magnetic stirrer, and refluxed 5.5 hours.Eliminate temperature, and (≤35 ℃) filter suspension when temperature still.With the other methanol rinse solid salt of about 10mL, concentrate the deep green filtrate that merges, produce the yellow-green colour solid of 21g humidity.

This solid is dissolved in the 2-propyl alcohol that 350mL boils, makes it crystallization at room temperature.With the crystallization that about 15mL 2-propyl alcohol and the rinsing of 15mL hexane produce, air-dry then, produce 15.5g glassy yellow product: N-(acridine-9-yl) Beta-alanine methyl ester hydrochloride (output is 78.5%).

¹H NMR: δ 1.9 (brs, 2H); 3.24 (t, J=7.0Hz, 2H); 3.76 (s, 3H); 4.45 (brs, 2H); 7.23 (app.t, J=8Hz, 2H); 7.49 (app.t, J=8Hz, 2H); 8.11 (d, J=8.4Hz, 2H); 8.30 (d, J=8.4Hz, 2H); 9.68 (brs, 0.5H) .IR:1574 (s), 1691 (s), 1726 (s), 2336 (m), 2361 (m), 3227 (m). two (2-hydroxyethyl) amino of step B.N-(acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride

At toluene (750mL), saturated water-based Na ₂CO ₃(200mL) and H ₂Distribute N-(acridine-9-yl) the Beta-alanine methyl ester hydrochloride that obtains by steps A between the O (50mL).(3 * 250mL) aqueous layer extracted once more merge organic layer and with saturated water-based Na with toluene ₂CO ₃(50mL) washing.Make the volume of toluene reduce to about 100mL by rotary evaporation.Add trolamine (30mL) then and form the immiscible system of a kind of part.The MeOH solution (2mL) that adds NaOMe (50mg) then.Quick removing from reaction mixture desolvated by stirring at room and rotary evaporation.Except that after desolvating, will place 1-1.5 hour in addition under the reaction mixture vacuum, produce syrup-like solution.

At CH ₂Cl ₂(200mL) and between the salt solution (200mL) distribute crude mixture, remove excessive trolamine.Use CH ₂Cl ₂(5 * 100mL) extracting brine layers.Merge organic layer,, use 0.5M HCl (2 * 100mL) extractings then with salt solution (50mL) washing.Merge acid water layer and use CH ₂Cl ₂(50mL) washing.At CH ₂Cl ₂(200mL) powder K is used in existence down ₂CO ₃Make acid solution become alkalescence.Separate organic layer, use CH ₂Cl ₂(5 * 100mL) extracting water layers once more.With the organic layer that salt solution (50mL) washing merges, use anhydrous Na ₂SO ₄Drying, and desorb is not contained the thick amine (5.02g) of glycol, a kind of yellow viscose glue.According to NMR, identical among this material and the embodiment 1 with the material that is equipped with the selection method preparation.

With the part (0.400g) of above-mentioned raw product and Virahol (100mL) vigorous stirring, and with the acidifying of 1MHCl diethyl ether solution.Cool slurry discards first batch of precipitation.After removing half solvent, second group of crystallization produces two (2-hydroxyethyl) amino of N-(acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride, for a kind of bright xanchromatic crystalline state solid (0.200g), according to HPLC purity＞95%.

¹H NMR: δ 8.11 (apparent t, 4H), 7.69 (apparent t, 2H), 7.41 (apparent t, 2H), 4.23 (t, J=5.4Hz, 2H), 4.03 (t, J=5.9Hz, 2H), 3.58 (t, J=5.2Hz, 4H), 2.73 (t, J=5.4Hz, 2H), 2.70 (t, J=5.9Hz, 2H) 2.68 (t, J=5.2Hz, 4H). do not find amine and hydroxyl proton.

¹³C NMR: δ 173.3,151.7,149.4,130.5,129.5,124.0,123.4,118.4,63.5,60.1,57.3,54.0,46.6, two (2-chloroethyl) amino of 35.8. step C.N-(acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride

To two (2-hydroxyethyl) amino of N-(acridine-9-yl) the Beta-alanine 2-[of step B acquisition] ethyl ester dihydrochloride (113mg, CH 0.24mmol) ₃(stirring suspension) adds SOCl in the CN solution (0.5ml) ₂(0.5mL).The yellow solution that stirring obtains under 23 ℃ 16 hours is removed volatile matter subsequently in a vacuum.Remaining orange oil be dissolved in EtOH (～2mL) in, remove EtOH in a vacuum, obtain a kind of yellow solid.(2 * 3mL) grind this material to use hexane then.Remove residual solvent in a vacuum, obtain two (2-chloroethyl) amino of the material that 123mg wishes: N-(acridine-9-yl) Beta-alanine 2-[] ethyl ester dihydrochloride (according to HPLC, purity is 93%), be a kind of yellow solid. ¹H NMR: δ 8.09 (apparent t, J=8.8Hz, 4H), 7.66 (apparent t, J=7.6Hz, 2H), 7.38 (apparent t, J=7.7Hz, 2H), 4.14 (t, J=5.9Hz, 2H), 4.00 (t, J=5.8Hz, 2H), 3.43 (t, J=6.9Hz, 4H), 2.87 (t, J=6.9Hz, 4H), 2.77 (t, J=5.9Hz, 2H), 2.69 (t, J=5.8Hz, 2H). do not find the amine proton. ¹³C NMR: δ 173.0,151.5,149.4,130.5, and 129.6,124.1,123.4,118.6,63.5,57.3,53.5,46.7,42.5,35.7.IR (the KBr precipitation of HCl salt): 3423,3236,2939,2879,1736,1634,1586,1572,1540,1473,1272,1173cm ^-1.

Embodiment 10

Two (2-chloroethyl) amino of N-(4-methoxyl group-acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride, Compound I X.

The preparation method of N-(4-methoxyl group-acridine-9-yl) Beta-alanine methyl esters is with 1.4g (5.84mmol) 4,9-dimethoxy acridine, 0.89g (6.42mmol) Beta-alanine methyl ester hydrochloride and 20mL methanol mixed, and heating then is at N ₂Under refluxed 12 hours.Concentrate this reactant then in a vacuum, be dissolved in CHCl ₃(50mL, 4: 1v/v), and use 50%NH in-the Virahol ₄OH (2 * 25mL) and salt solution (1 * 25mL) washing.Use Na ₂SO ₄Dry organic layer concentrates in a vacuum and produces 1.24g (68%) methyl esters (according to HPLC, purity＞74%), is xanchromatic oil; R _f(SiO ₂, ethyl acetate)=0.25; IR (film): 3363,2947,1730,1611,1573,1518,1484,1463,1423,1420,1246,1170,1081cm ^{-1 1}H NMR: δ 2.70 (t, 2H, J=5.7Hz), 3.74 (s, 3H), 4.00 (t, 2H, J=6.3Hz), 4.11 (s, 3H), 6.98 (d, 1H, J=7.4Hz), 7.36 (m, 2H), 7.65 (m, 2H), 8.12 (d, 2H, J=8.5Hz); ¹³C NMR): δ 35.7,46.9,52.3,56.5,107.2,115.3,119.8,123.5,124.1,130.0,151.4,173.6.

Under the described condition of embodiment 3 step B, be translated into glycol, produce the 647g yellow oil.The HPLC of crude mixture analyzed show 85% productive rate (λ=278nm); R _f(SiO ₂, 20% methyl alcohol-ethyl acetate)=0.17; IR (film): 3337,2947,2828,1726,1616,1569,1522,1484,1463,1420,1348,1250,1174,1127,1081,1043cm ^{-1 1}H NMR: δ 2.7 (m, 8H), 3.55 (m, 4H), 3.97-4.08 (m, 2H), 4.08 (s, 3H), 4.19 (t, 2H, J=5.5 Hz), 6.96 (d, 1H, J=7.4Hz), 7.29 (m, 2H), 7.61 (m, 2H), 8.10 (m, 2H); ¹³C NMR: δ 36.0,46.9,53.7,56.4,57.1,60.1,63.3,107.4,115.7,119.1,119.6,123.2,123.5,123.9,128.5,130.0,140.8,147.4,151.6,151.7,154.3,173.3.

As described in embodiment 3 step C, be translated into two (2-chloroethyl) amino of N-(4-methoxyl group-acridine-9-yl) Beta-alanine 2-[] ethyl ester dihydrochloride and thionyl chloride.Use ethyl acetate with 10% methyl alcohol-ethyl acetate crude product to be filtered (SiO rapidly subsequently ₂), obtaining the 58mg yellow oil after the degraded on the obvious post of some product; R _f(SiO _Ux, ethyl acetate)=0.26; IR (film): 3405,2955,2828,1726,1616,1577,1518,1463,1416,1348,1246,1174,1123,1081,1013cm ^{-1 1}H NMR: δ 2.69-2.99 (m, 8H), 3.45 (t, 4H, J=6.7 Hz), 4.03 (m, 2H), 4.09 (s, 3H), 4.16 (t, 2H, J=5.9Hz), 6.97 (d, 1H, J=7.7Hz), 7.32 (m, 2H), 7.65 (m, 2H), 8.12 (d, 2H, J=8.7Hz).

Remove excessive thionyl chloride (2 * 5mL toluene) by concentration of reaction solution and azeotropic in a vacuum, can be with crude product isolated in form dihydrochloride.The starting material completely consumed of HPLC analysis revealed and 4-methoxyl group acridine (R _T=22.3 minutes) be main impurity. ¹H?NMR(CD ₃OD)：δ3.18(t，2H，J＝6.4Hz)，3.71(m，6H)，4.04(m，4H)，4.18(s，3H)，4.51(m，2H)，7.17(m，2H)，7.56(m，2H)，7.91-8.15(m，2H)，8.55(d，1H，J＝8.8Hz).

Similarly prepare two (2-chloroethyl) amino of N-(3-chloro-4-methylacridine-9-yl) Beta-alanine 2-[from 3-chloro-9-methoxyl group-4-methylacridine] the ethyl ester dihydrochloride, compounds X.Free alkali ¹HNMR: δ 7.96-8.17 (m, 3H), 7.29-7.52 (m, 3H), 4.19 (t, J=5.8Hz, 2H), 4.00 (s, 3H), 3.89 (t, J=5.1Hz, 2H), 3.47 (t, J=6.8Hz, 4H), 2.91 (t, J=6.8Hz, 4H), 2.83 (t, J=5.8Hz, 2H), 2.67 (t, J=5.5Hz, 2H).

Similarly from 6-chloro-2,9-dimethoxy acridine prepares two (2-chloroethyl) amino of N-(6-chloro-2-methoxyl group acridine-9-yl) Beta-alanine 2-[] the ethyl ester dihydrochloride, compounds X III.Free alkali ¹HNMR: δ 7.96-8.17 (m, 3H), 7.29-7.52 (m, 3H), 4.19 (t, J=5.8Hz, 2H), 4.00 (s, 3H), 3.89 (t, J=5.1Hz, 2H), 3.47 (t, J=6.8Hz, 4H), 2.91 (t, J=6.8Hz, 4H), 2.83 (t, J=5.8Hz, 2H), 2.67 (t, J=5.5Hz, 2H).

Embodiment 11

[N, N-two (2-chloroethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] the propyl ester dihydrochloride, compounds X I.Steps A. Beta-alanine [N, two (the 2-triisopropyl silyl oxygen base) ethyls of N-] ethyl ester

With the Beta-alanine carbethoxy hydrochloride (1.99g, 12.9mmol), K ₂CO ₃(6.0g is 43.4mmol) with iodine ethyl three different third silyl esters (9.47g, the slurry reflux 5-7 of acetonitrile solution 28.9mmol) (175mL) days.After the solvent vacuum-evaporation, use CH ₂Cl ₂Abrasive solid.Na with dilution ₂CO ₃(moisture) uses salt water washing organic layer then, in anhydrous Na ₂SO ₄Last dry.By silica gel column chromatography (1: 4 EtOAc/ hexane) purifying crude product, obtain 5.60g oily [N, two (the 2-triisopropyl siloxy-) ethyls of N-] Beta-alanine ethyl ester (83.1%). ¹H NMR: δ 4.12 (q, J=7.1Hz, 2H), 3.73 (t, J=6.8Hz, 4H), 2.92 (t, J=7.3Hz, 2H), 2.70 (t, J=6.6Hz, 4H), 2.46 (t, J=7.4Hz, 2H), 1.4-0.9 (m, 45H, the triplet when comprising 1.25 (3H) and 1.06 and 1.05 o'clock monomer) step B.[N, two (the 2-triisopropyl silyl oxygen base) ethyls of N-]-Beta-alanine

In ethanol, stir Beta-alanine [N, two (2-triisopropyl the siloxy-)-ethyls of the N-] ethyl ester that obtains by above-mentioned steps A (5.60g, 10.8mmol) and lithium hydroxide (0.59g 14.1mmol), and refluxed 3 hours.Remove and desolvate, at CH ₂Cl ₂NaHCO with dilution ₃Distribute crude product between (moisture).With salt water washing organic layer, in anhydrous Na ₂SO ₄Last dry, obtain [N, two (the 2-triisopropyl silyl oxygen base) ethyls of N-]-Beta-alanine after the desorb, be a kind of flaxen oil (5.03g, 95.1% output). ¹H NMR: δ 3.90 (t, J=5.5Hz, 4H), 3.04 (t, J=6.2Hz, 2H), 2.92 (t, J=5.5Hz, 4H), 2.50 (t, J=6.1Hz, 2H), 1.06 (s, 42H). step C.[N, N-two (2-hydroxyethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] propyl ester

In N ₂Down at CH ₂Cl ₂[N, two (the 2-triisopropyl siloxy-) ethyls of N-]-Beta-alanine that stirring is obtained by above step B (1mL) (51.0mg, 0.104mmol).When on ice bath, cooling off, dropwise add SOCl ₂(0.5mL), and stirring reaction liquid 2.25 hours.The desorb reaction mixture is removed excessive SOCl ₂After, add exsiccant CH ₂Cl ₂(0.5mL), and in N ₂In ice bath, cool off this solution down.9-(3-hydroxyl) Propylamino-6-chloro-2-methoxyl group-acridine (29.0mg, 91.5mmol) CH that adds precooling ₂Cl ₂Slurries (1mL).0.5 after hour, at CH ₂Cl ₂With moisture NaHCO ₃Between distribute this mixture.With salt water washing organic layer, with anhydrous Na ₂SO ₄Last dry and desorb.Grind the glue that obtains with hexane, the desorb hexane extract obtains starting material and mixture of products (53.5mg) that a kind of extremely thick triisopropyl silyl is protected.

In order to remove three different third silyls, in ice-cold THF (1mL), stir the thick glycol (33.1mg) of part protection.After adding HF/ pyridine (0.5ml), be full of N ₂Air bag in stirred the mixture at ambient temperature 2.5 hours.At CH ₂Cl ₂With NaHCO ₃Distribute reaction mixture between (moisture), with the NaHCO of dilution ₃(moisture) washing organic layer is removed excessive HF/ pyridine for several times.With using anhydrous Na behind the salt solution preliminarily dried ₂SO ₄Drying, removing desolvates obtains thick glycol (13.1mg).

Itself and other thick glycol (5.0mg) are merged, by with 95CH ₂Cl ₂/ 5iPA/1 TFA prepares type TLC purifying as the C-18 of elutriant, obtains the glycol tfa salt.With salt at CH ₂Cl ₂With NaHCO ₃After distributing between (moisture),, use anhydrous Na then with the dry organic layer of salt solution ₂SO ₄Drying, and desorb obtains the free alkali of glycol, [N, N-two (2-hydroxyethyl)] Beta-alanine, 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] propyl ester (5.0mg). ¹H NMR: δ 7.92-8.25 (m, 3H), 7.23-7.47 (m, 3H), 430 (t, J=5.7Hz, 2H)), 3.98 (s, 3H), 3.81 (t, J=6.2Hz, 2H), 3.64 (t, J=4.9Hz, 4H), 2.86 (t, J=6.1Hz, 2H), 2.67 (t, J=4.9Hz, 4H), 2.51 (t, J=5.9Hz, 2H), 2.04 (tangible 5-linked body, 2H) step D.[N, N-two (2-chloroethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] the propyl ester dihydrochloride, chemical combination material XI

[N, N-two (2-hydroxyethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl with above acquisition) amino] (4.0mg 0.0073mmol) is dissolved in CH to propyl ester ₂Cl ₂(1mL), in ice/water-bath, cool off.Add ice-cold SOCl ₂(0.1mL), reaction solution was at room temperature stirred 4 hours.The desorb reaction mixture removes and desolvates, grinds with hexane, and at CH ₂Cl ₂With NaHCO ₃Distribute between (moisture).With the dry organic layer of salt solution, use anhydrous Na then ₂SO ₄Dry also desorb obtains the dichloro compound into a kind of yellow glue. ¹H NMR: δ 7.8-8.2 (m, 3H), 7.2-7.5 (m, 3H), 4.35 (t, J=5.9Hz, 2H), 3.85-4.10 (3.99, s, OMe and 3.9-4.0, m, NHCH ₂, total 5H), 3.48 (t, J=6.9Hz, 4H), 2.9-3.0 (m, 6H), 2.49 (t, J=6.6Hz, 2H), 2.1-2.3 (m, 2H).

CH in precooling ₂Cl ₂The middle unhindered amina that stirs, with the acidifying of 1M HCl diethyl ether solution, and with the desorb of a few drops methyl alcohol, obtain the compound of hope, [N, N-two (2-chloroethyl)] Beta-alanine 3-[(6-chloro-2-methoxyl group acridine-9-yl) amino] propyl ester dihydrochloride (2.5mg), (3.5mg, 81%), is a kind of yellow solid.

With the method identical, but use 6-chloro-9-(2-hydroxyl) ethylamino--2-methoxyl group-acridine to replace 6-chloro-9-(3-hydroxyl) Propylamino-2-methoxyl group-acridine, prepare similar glycol with abovementioned steps C. ¹H?NMR：δ7.96-8.13(m，3H)，7.20-7.47(m，3H)，4.76(t，J＝4.9Hz，2H)，3.99(s，3H)，3.92-4.14(m，2H)，3.60(t，J＝5.1Hz，4H)，2.78(t，J＝6.1Hz，2H)，2.63(t，J＝5.1Hz，4H)，2.45(t，J＝6.0Hz，2H).

By being similar to the step of step D, it is converted into [N, N-two (2-chloroethyl)] Beta-alanine 2-[(6-chloro-2-methoxyl group acridine-9-yl) amino] the ethyl ester dihydrochloride, compounds X II. ¹H?NMR：δ7.94-8.20(m)，7.20-7.50(m)，4.42(CH ₂OC＝O)，3.90-4.10(OCH ₃，NHCH ₂)，3.46(CH ₂Cl)，2.82(N(CH ₂) ₃)，2.39-2.56(CH ₂C＝O).

Embodiment 12

4,5 ', 8-trimethylammonium-4 '-psoralene acetic ester hydrochloride, compounds X IV steps A .4,5 ', 8-trimethylammonium-4 '-psoralene acetate [N, N-two (2-hydroxyethyl)]-2-aminoethyl ester

Down stir 4,5 ' at 100 ℃, and 8-trimethylammonium-4 '-psoralene methyl acetate (250mg, 0.832mmol), the HCl (2mL) in trolamine (12mL) and the 1M ether 2 hours.Make the clarification brown solution that obtains be cooled to room temperature, and at CH ₂Cl ₂With saturated NaHCO ₃Distribute between (moisture).Use saturated NaHCO ₃(moisture) rinsing organic layer for several times.Use anhydrous Na ₂SO ₄After the drying, remove solvent in a vacuum, at CH ₂Cl ₂And distribute resistates between the moisture HCl of 1M.Use CH ₂Cl ₂The rinse water number of plies is inferior, uses K then in the presence of organic solvent ₂CO ₃Make it to become alkalescence.The organic layer that contains neutral products with water rinse for several times, and is dry then and concentrate.The repetition of soda acid extractive process can produce a kind of beige solid and wish product (84.3mg, 24.3%): ¹HNMR: δ 7.53 (s, 1H), 6.24 (s, 1H), 4.23 (t, J=5.4Hz, 2H), 3.69 (s, 2H), 3.56 (t, J=5.3Hz, 4H), 2.82 (t, J=5.4Hz, 2H), 2.69 (t, J=5.3Hz, 4H), 2.57 (s, 3H), 2.51 (d, J=1.1Hz, 3H), 2.47 (s, 3H). step B.4,5 ', 8-trimethylammonium-4 '-psoralene acetate [N, N-two (2-chloroethyl)]-2-aminoethyl ester hydrochloride

To ice-cold above-mentioned glycol (9.8mg, 0.023mmol) CH ₂Cl ₂Add thionyl chloride (0.2mL) in the mixed solution (1mL), and under nitrogen, cross liquid in stirring at room.Concentrate the slurries that obtain, grind with hexane then, obtain hope product (6.2mg, 53.9%) into a kind of pale solid: ¹H NMR (CD ₃OD): δ 7.71 (s, 1H), 6.28 (s, 1H), 4.56 (t, J=4.8Hz, 2H), 3.95 (t, J=6.1Hz, 4H), 3.89 (s, 2H), 3.60-3.83 (m, 6H), 2.54 (s, 3H), 2.53 (s, 3H), 2.50 (s, 3H).

Embodiment 13

Typical case BMT a small amount of or little transplanting scheme

Before with the allosome autologous peripheral blood stemcell transplant, implement a kind of non-isolating counterplan for the patient who shows CLL.Support with DLI after transplanting that this is to reach maximum graft anti-malignant tumor to react necessary.Slight toxic counterplan is based on fludarabine (FAMP), and is improved according to the malignant tumour type.For late period and previously treated CLL, the patient is used the FAMP of 300mg/m2/dx3 and the endoxan of 30mg/m2/dx3.The scheme of using the Ara-c by the cis-platinum of FAMP, the 25mg/m2/d/CIx4 of 30mg/m2/dx3 and 0.5gm mg/m2/dx2 to form is handled Richter and large celllymphoma (LCL).Except LCL is handled, do not carry out the GVHD prevention.This scheme is in order to produce chimerism, and makes to implant and become possibility, and reduces the toxicity of conventional inductive treatment.Can impose DLI after transplanting 1 month strengthens transplanting.Successful transplanting has been done preparation for the DLI that uses later on, is necessary for strengthening the reaction of graft leukemia and quickening immune restoration.

Embodiment 14

Use the in vitro study of human peripheral lymphocyte

Stand PCT and in the several characteristic of the human peripheral lymphocyte (PBL) of vitro culture in order to characterize the processed cell colony of particular type, to have checked.These comprise viability, T cell proliferation, cytokine is synthetic and secretion, surface antigen expression, vitro cytotoxicity and Fas ligand expression.Viability

Be determined at 3J/cm with trypanblue exclusion method ²Accept the viability of the PBL of 10nM S-59 under the UVA.Survival rate is to be to be 25% after 50%, 4 day after 75%, 3 day after 2 days.In relevant experiment, observe the survival rate of processed lymphocytic dependent dose.Propagation

According to ³H-thymidine mixing in the T cell DNA that allosome stimulates measured lymphocytic propagation.Separating periphery blood monocytic cell (PBMC) from three donors merges, and gamma-irradiation (2500cGy) preventing autostimulation, and is used as irritation cell in MLR.Isolating PBMC action effect cell from the 4th donor.The effector cell is at 3J/cm ²UVA is down with 0.05,0.5 or 5nM 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen (" S-59 ") is handled, and with the collaborative cultivation of irritation cell.Contrast, untreated effector cell also cultivate with irritation cell is collaborative.Cultivate after 6 days, in nutrient solution, add ³The H-thymidine, harvested cell after a day is by scintillation counting analysis of cells sample determination ³The absorption of H-thymidine.Fig. 8 shows, according among the MLR ³The H-thymidine is taken in the processed leukocytic propagation of being measured, and is eliminated in dose-dependent mode between 0.1nM-10nMS-59, and propagation is suppressed fully during 10nM S-59.

Also available a kind of selection method that is equipped with is analyzed processed leukocytic multiplication capacity, and the anti-CD 3 antibodies with surface bonding in this method activates processed cell.In this test, PBMC or not processed or at 3J/cm ²UVA dosage is handled with 0.0001,0.001 or 0.01 μ M S-59 down or is shone when no S-59.Then in adhering to the culture plate of anti-CD 3 antibodies at 5%CO ₂, 37 ℃ of following incubation cells, come the polyclone of inducing T cell to activate, and after cultivating 1,2 and 3 day, measure ³Mixing of H-thymidine.The inhibition fully that result (Fig. 9) breeds when being presented at the reduction of multiplication capacity dependent dose under all treatment condition and 10nM S-59.Cytokine produces

As preceding for described in the part of proliferation assay, cultivator lymphocyte in MLR.At 3.0J/cm ²UVA is down with the S-59 of different concns (0.05,0.5 or 5nM) Treatment Effects cell.Cultivate and take MLR cell culture medium sample after 2,4,6 and 7 days.Measure the IL-2 and the IFN-γ level of these samples with sandwich enzyme-linked immunosorbent adsorption test, and use the spectrophotometry quantitative result.Standardized solution is used for setting up the typical curve that concentration (pg/ml) is associated with absorbancy, comes cytokine concentration in the confirmed test sample by it.

Figure 10 A shows the analysis that IL-2 is produced.Untreated lymphocyte and reach the highest with the lymphocytic IL-2 secretion that low concentration S-59 (that is, 0.005 and 0.5nM) handles when cultivating 2 days is then because IL-2 is bred lymphocyte consumption in the colony descend.On the contrary, in the lymphocyte of handling with 5nM S-59, IL-2 is by the lymphocyte consumption of non-propagation, and still is present in the substratum with quite high level in the time of the 4th, 6 and 7 day.

The IFN-γ of stimulation PBMC produces the influence that is not subjected to the S-59+UVA processing or is improved slightly.See Figure 10 B.The synthesis result of IL-2 and IFN-γ shows that under the repressed condition of propagation, processed cell still can synthesize justacrine and t cell activation related cytokine.Surface antigen is expressed

Analyzed the analysis of processed lymphocyte upper surface marker expression.Obtain lymphocyte through the Ficoll gradient centrifugation, and activate by anti-CD 3 antibodies stimulated in vitro with surface bonding.Use the fluorescence antibody of CD69, CD25 (IL-2 acceptor) or CD40L, express by the lymphocytic surface antigen that fluorescence-activated cell sorting (FACS) assay determination is activated, handles.Express as the function mensuration that activates the back time, and compare with the expression of untreated cell.

CD69 is a kind of early stage mark of lymphocyte activator.Figure 11 A shows, with 1nM or 10nM S-59+3J/cm ²The cell that UVA handles is similar to or a little more than the surface C D69 level of untreated cell up to having in 48 hours time after processing.Therefore, although processed cell can not be bred, still tool is functional with activating relevant signal pipeline.

CD25 is the IL-2 acceptor, and slightly evening, (activating the back) detected its expression in CD69.Figure 11 B shows, compares with untreated cell, with 1nM or 10nM S-59+3J/cm ²In the cell that UVA handles, surface C D25 is unaffected basically.

The CD40 molecule of expressing on CD40 part (CD40L) the identification B cell surface on the T cell is as the part of t cell activation process.The inhibition that CD40L expresses on the T cell can inducing cell unresponsiveness or anergy.Therefore, handling back CD40L undisturbed expression on the T cell is the indication of normal t cell activation.Make with anti-CD 3 antibodies and to check the influence (Figure 12) that PCT expresses CD40L behind the treated cell-stimulating.The result of this analysis shows, for the treatment condition that multiplication capacity subsequently is eliminated, the CD40L of processed cell expresses and observed close parallel in untreated cell.

In a word, under the serious condition that reduces or suppress fully of propagation (seeing Fig. 8 and 9, present embodiment), the expression of earlier T cell-stimulating mark CD69 and IL-2 acceptor CD25 is still unaffected basically; And the CD40L level is parallel with untreated cell.Should be pointed out that CD69 i.e. transient expression on lymphocyte in case through antigen or mitogenesis stimulation, the continuation of CD69 is expressed needs new transcribing.Therefore, observe high CD69 level in treated non-proliferative cell, this has further supported to handle reversibly this opinion of inhibition of gene expression.The cytotoxic T cell function

By in MLR, producing cytotoxic T cell, estimate that S-59+UVA handles the influence to T lysis function to allogeneic " stimulation " cell of gamma-radiation deactivation.Measure these cytotoxic T cell cracking then ⁵¹The ability of the target cell of Cr-mark, these target cells obtain from the identical donor of the irritation cell that is used to provide deactivation.

The generation of cytotoxic T cell

Take out peripheral blood (100mL) from two kinds of donors, a kind of being called as " stimulator ", another kind is called as " effector ".From two kind of groups, separate PBMC through the Ficoll gradient centrifugation.By with effect PBMC with 5 * 10 ⁶The cell density of cell/ml placed tissue culture flasks 1 hour, consumed the monocyte among the poor effect PBMC.Gamma-irradiation (2500cGy) stimulates PBMC blocking-up cell fission.The stimulator that monocyte is consumed poor effector and irradiation separates and is resuspended in the RPMI perfect medium to 1 * 10 ⁶The concentration of cell/ml.Merge isopyknic effector then and the irritation cell suspension is used for MLR, and at 5%CO ₂37 ℃ of incubations are 7 days in the incubator.

The preparation of target cell

From the identical donor that irritation cell is provided for MLR, extract peripheral blood (50ml).As above separate PBMC, with 1 * 10 ⁶The concentration of cell/ml is resuspended in the RPMI substratum, and (phytohemagglutinin-M) recombinant il-2 with 10 units/ml stimulated 7 days down at 37 ℃ to use 2 μ g/ml PHA-M then.

With ⁵¹Cr is to the mark of target cell

At 200 μ Ci Na ₂ ⁵¹CrO ₄There are following 37 ℃ of incubation target cells 2 hours, wash three times then and remove uncorporated marker.

The photochemical treatment of pairing effect cell (PCT)

Effector cell's (monocyte consumption poor) as mentioned above is divided into four equal portions, each part is resuspended among the PBS that 30ml contains 1% bovine serum albumin.The final concentration that in three parts, adds S-59 to 0.1,0.01 or 0.001 μ M; All the other equal portions are as untreated contrast.The sample transfer that will contain S-59 is used 3J/cm in Baxter/Fenwall UVA irradiating unit to 30ml 2410 blood bags ²UVA shines each sample.

CTL measures

Cytotoxic T lymphocyte (CTL) activity of the effector cell colony that following mensuration is processed.The processed cell of resuspension (or contrast untreated cell) is to the 1ml final volume 5 * 10 ⁶The final concentration of cell/ml carries out continuous twice dilution and obtains 2.5 * 10 ⁶, 1.25 * 10 ⁶With 0.625 * 10 ⁶The final concentration of cell/ml.In every hole in three holes of round bottom 96 hole microtiter plates, add the treated effector cell of resuspension and 100 μ l samples of each treated effector cell's extent of dilution (" effector cell ").In every effect of holes cell, add then and contain 5 * 10 ³100 μ l labels targets cell samples of individual cell (preparation) as mentioned above produce the effector cell of 100: 1,50: 1,25: 1 and 12.5: 1: target cell ratio, and in 37 ℃ of following incubation mixtures 4 hours.Behind the incubation,, from every hole, take out 100 μ l supernatant liquors with the centrifugal culture plate of 250 * g 5 minutes.On gamma counter quantitatively in the supernatant liquor ⁵¹The amount of Cr.Every group of three hole calculated average ⁵¹Cr cpm and standard deviation.

Contrast

Dilute unlabelled target cell, and, measure with the target cell splice of the ratio identical with mark with the effector cell ⁵¹The spontaneous release of Cr.The target cell of mark also with RPMI and 1%Triton X-1000 (each 3 hole) incubation, is measured ⁵¹The maximum of Cr discharges.Every group of three hole calculated average ⁵¹Cr cpm and standard deviation.

The result

Figure 13 shows for treated and undressed white corpuscle, at the different effect thing: target (or unlabelled target: the target of mark) during ratio ⁵¹Cr discharges.These results show that the white corpuscle of handling with the S-59 dosage that can block propagation and UVA has kept molten cell function.Therefore, the processed white corpuscle that can not breed not only has the phenotype identical with untreated cell (shown in surface markers and cytokine-expressing), and has similar functional property.

In a word, to the propagation of processed leukocyte population, surface markers express, cytokine is synthetic and the active analysis of CTL has shown the following properties of processed cell:

1. in a single day treated, promptly can be observed the dose-dependent inhibition of multiplication capacity.

2. processed cell reservation cytokine is synthetic, cell-surface antigens is expressed and vitro cytotoxicity.

Therefore, the leukocyte population of said processing and suitable cell mass physical efficiency help the implantation of donorcells (as hematopoietic cell and hemopoietic stem cell) in the myelosuppressive host of non-severe.A kind of mechanism that can realize this point is by treated leukocyte population inducing the special tolerance of donor.

Embodiment 15

The preparation of processed white corpuscle GVL effect when no GVHD in the mouse model system

The research S-59+UVA that experimentizes handles the active influence of donor lymphocyte GVL in the B6/AKR mouse model system of MHC-mispairing.Make donor spleen cells pass through fine mesh screen, obtain the single cell suspension of unsegregated splenocyte, the UVA with 0.01 μ M S-59 and various dose handles (0.5 minute, 1 minute, 2 minutes and 8 minutes) then.With 1 * 10 ⁷The donor spleen cells of individual processing and 5 * 10 ⁶The medullary cell of individual eliminating T cell is (C57BL/6, H-2b, Thy1.2 together ⁺) be injected into AKR, the H-2k, the Thy1.1 that stand the 1100R total body radiation ⁺Among the host.Transplant after 3 days and attack every group of mouse (every group of 6 animals) with 250 AKR-M2 leukemia cells.Observe GVHD clinical symptom, leukemia relapse and the death of transplanting animal.In addition, transplant the back and write down the body weight of once transplanting animal in every 3-4 days.

The results are summarized in table 3 and Figure 14 and 15, show the proper range that can obtain UVA+S-59 dosage, it can keep the GVL activity effectively and reduce GVHD in the MHC-mispairing is transplanted.For example, (the 0.5 minute UVA+0.01 μ M S-59 that in the marrow of getting rid of the T cell, adds untreated or gentle processing; Irradiation dose=1.8 J-nM/cm ²) lymphocyte causes slightly (proving according to the body weight that reduces in these treated animals to severe GVHD in transplant recipient; Figure 14).On the contrary, with 1 minute UVA+0.01 μ M S-59 (irradiation dose=3.7 J-nM/cm ²) lymphocyte handled can keep GVL activity (leukemia was attacked after 70 days, 3 merely hit 3 survivors) effectively and not have the GVHD symptom.See Table 3 and Figure 15.The body weight (Figure 14) of the animal of handling with 1 minute UVA+0.01 μ M S-59 also shows no GVHD.If (2 and 8 minutes UVA are equivalent to 7.5 and 30 J-nM/cm respectively to reach higher UVA dosage with identical S-59 concentration ²Irradiation dose) handle cell, then the GVL of donor lymphocyte is active eliminates.In these cases, transplanting in back 20 days has 3 death in 3 mouse, sees Figure 15.

Table 3

Use the GVL activity in the leukocytic B6/AKR mosaic of PCT

			Leukemia is attacked		Healthy state
						Group	Handle		Do not have	250 cells
????1	0.01 8 minutes UVA of μ M S-59	# survival: MST: scope:	?3/3 ?＞70 ?X，X，X	????1/3 ????18 ????18，18，19							Survivor's health
					????2	0.01 2 minutes UVA of μ M S-59	# survival: MST: scope:	?3/3 ?＞70 ?X，X，X	????1/3 ????18 ????18，18，21	Survivor's health
????3	0.01 1 minute UVA of μ M S-59	# survival: MST: scope:	?3/3 ?＞70 ?X，X，X	????3/3 ????＞70 ????X，X，X							Healthy: can not distinguish that leukemia attacks with the mouse of not attacking
					????4	0.01 0.5 minute UVA of μ M S-59	# survival: MST: scope:	?2/3 ?＞70 ?58，X，X	????3/3 ????＞70 ????X，X，X	In the mouse that leukemia is attacked and do not attacked, severe GVHD is arranged all
????5	0.01 μ M S-59 does not have UVA	# survival: MST: scope:	?3/3 ?＞70 ?X，X，X	????3/3 ????＞70 ????X，X，X							Moderate GVHD
					????6	BMT only	# survival: MST: scope:	?4/4 ?＞70 ?X，X，X，X	????0/5 ????18 ????17，18，18，18，19	Healthy

Attention :-all groups were all shone at the 1st day and are accepted the bone marrow transplantation of MHC-mispairing and the 0th day

Treated splenocyte (as being suitable for).

If-carry out leukemia to attack, then at the 3rd day.

The intermediate value of-MST=survival time, unit is the sky.

-scope=dead fate (transplanting the back) (X=survival)

Can reach a conclusion from these results, adding the white corpuscle of handling with an amount of S-59+UVA in the marrow of getting rid of the T cell can prevent GVHD in main MHC-mispairing mouse model system.In addition, suitably the leukocytic adding of handling helps the donor implantation, and causes the stable chimerism among the MHC-mispairing transplanting host.At last, processed leukocytic GVL is active keeps.Therefore, be that a kind of immunologic competence of donor lymphocyte of regulating is to support the novel method of allotransplantation with S-59+UVA to leukocytic processing.

Embodiment 16

S-303 is to the influence of human lymphocyte propagation and T cell function

In the MLR test, stimulate the T cell to measure to treated allosome.From in order to prevent autostimulation with separating periphery blood monocytic cell (PBMC) the gamma-emitting donor of 2500cGy dosage, as irritation cell.Be used as the effector cell from the isolating PBMC of different donors.With two (2-chloroethyl) amino of N-(acridine-9-yl) the Beta-alanine 2-[of 0.1,0.2,0.3,0.4 or 0.5 μ M] ethyl ester (S-303) at room temperature concentration of treatment be 2 * 10 ⁶About 15 minutes of the effector cell of cell/ml washes twice with the PBS that contains 1% bovine serum albumin.That S-303 is handled and untreated effector cell and γ-radiating irritation cell are with the collaborative cultivation of 1: 1 ratio.

Lymphopoietic measuring method comprises that collaborative the cultivation begins to add after 6 days in collaborative culture ³The H-thymidine in the 7th day harvested cell, and is measured in the cell ³H cpm mixes.The culture supernatant that collaborative cultivation beginning was taked after 24 and 48 hours is carried out sandwich ELISA, the generation of the analysis of cells factor.

The analysis (Figure 16) of S-303 being handled the multiplication capacity of cell shows, in the allosome stimulatory effect cell that S-303 handles ³Mixing of H-thymidine reduced in the cell of handling with 0.1 μ M S-303, and is blocked fully in the cell that with concentration is 0.2 μ M or higher S-303 processing.Therefore, in the white corpuscle that S-303 handles, observe lymphopoietic dose-dependent inhibition.

As S-303 being handled the mensuration that leukocytic cytokine produces, estimate the generation of IFN-(IFN-γ).Figure 17 shows that in the cell of handling with 0.1 μ M and 0.2 μ M S-303, IFN-γ secretion is unaffected, wherein breeds serious respectively the reduction and inhibition (seeing Figure 16) fully.With the cell that 0.3 μ M S-303 handles, wherein lymphocytic propagation is suppressed (Figure 16) fully, produces 28% about IFN-γ (Figure 17) of control level.

In a word, the S-303 pre-treatment suppresses the propagation of the human lymphocyte that allosome stimulates in dose-dependent mode, as measuring in MLR.In addition, under the condition that propagation is suppressed fully (for example, with the processing of 0.2 μ M S-303 pairing effect cell), IFN-γ's is synthetic unaffected.Therefore, can obtain to be created in and to breed after the stimulation but keep the lymphocytic S-303 treatment condition of immunologic competence.

Although for the clarity of understanding, described in detail foregoing invention in the mode of chart and embodiment, those skilled in the art should be understood that under the situation that does not deviate from spirit of the present invention can carry out multiple change and modification.Therefore, should not regard foregoing description and embodiment as and limit the scope of the invention.

Claims (98)

1. one kind is ready to use in the isolated cells colony that introduces in the allogeneic acceptor, contain a kind of leukocyte population in this cell colony, wherein the part of this leukocyte population is not bred, make that inducing of graft versus host disease (GVHD) is suppressed in this receptor, and wherein the part of this leukocyte population keeps immunologic competence.

2. the cell colony of claim 1, wherein leukocyte population contains T cell, NK cell and antigen presenting cell.

3. the cell colony of claim 1, wherein the part of this leukocyte population promptly produces cytokine after suitably stimulating.

4. the cell colony of claim 3, wherein cytokine is selected from: IL-1, IL-2, IL-4, IFN-γ, IL-10 and GM-CSF.

5. the cell colony of claim 1, wherein the part of this leukocyte population is expressed a kind of surface markers, and this surface markers is selected from: CD2, CD28, CTLA4, CD40 part (gp39), CD18, CD25, CD69 (lymphocyte activator mark) and CD16/CD56, MHC I class and II class, CD8, CD4, CD3/TcR (TXi Baoshouti), CD54 (ICAM-1), LFA-1 and VLA-4.

6. the cell colony of claim 1 wherein surpasses 90% white corpuscle and does not breed in this colony.

7. the cell colony of claim 1 wherein stimulates white corpuscle by contact antigen or mitogen.

8. the cell colony of claim 1, wherein white corpuscle is effective aspect the destruction that promotes the disease cell.

9. the cell colony of claim 1, wherein white corpuscle is effective aspect the destruction that promotes infected cell.

10. the cell colony of claim 1, wherein white corpuscle helps the implantation of second kind of cell colony.

11. the cell colony of claim 10, wherein second kind of cell colony is selected from: hematopoietic cell, myelocyte, white corpuscle, medullary cell, islet cells, liver cell, neuronal cell, myocardial cell, mesenchymal cell and endotheliocyte.

12. the cell colony of claim 10, wherein second kind of cell colony is a kind of organ.

13. the colony of claim 1, wherein this leukocyte population contains first kind of lymphocyte subgroup.

14. the colony of claim 13, wherein first kind of lymphocyte subgroup contains second kind of t lymphocyte subset group.

15. the colony of claim 14, wherein the expression according to surface markers obtains second kind of subgroup.

16. the colony of claim 15, wherein surface markers is selected from CD8, CD4, CD16 and CD56.

17. the colony of claim 1 wherein obtains leukocyte population by the method that is selected from the removal of white corpuscle electrophoresis and red corpuscle from whole blood.

18. a method that is used for donor leukocyte infusion, wherein this method is included in the cell colony of introducing after the bone marrow transplantation according to claim 1 in being tried Mammals.

19. a method that strengthens the implantation of transplanted cells colony is introduced the cell colony according to claim 1 in being tried Mammals before wherein this method is included in and transplants.

20. a method that strengthens the implantation of transplanted cells colony, wherein this method is included in the cell colony of introducing when transplanting according to claim 1 in being tried Mammals.

21. a method that strengthens the implantation of transplanted cells colony is introduced the cell colony according to claim 1 in being tried Mammals after wherein this method is included in and transplants.

22. according to the method for claim 20, wherein the cell colony of Yi Zhiing contains hemopoietic stem cell.

23. according to the method for claim 21, wherein the cell colony of Yi Zhiing contains hemopoietic stem cell.

24. a method that is used for immunologic reconstitution, wherein this method comprises the cell colony of introducing according to claim 1 in being tried Mammals.

25. a method that is used for adoptive immunotherapy, wherein this method comprises the cell colony of introducing according to claim 1 in being tried Mammals.

26. a method that is used to mix the chimerism treatment, wherein this method comprises the cell colony of introducing according to claim 1 in being tried Mammals.

27. a method for preparing according to the cell colony of claim 1, wherein this method comprises:

(a) form a kind of vitro reactions mixture, it contains leukocyte population and a kind of compound that can form covalent linkage with nucleic acid; With

(b) this reaction mixture of incubation under the condition that can produce a kind of cell colony that contains leukocyte population;

Wherein a part of leukocyte population is not bred, and makes that inducing of graft versus host disease (GVHD) is suppressed in the acceptor, and wherein the part of this leukocyte population keeps immunologic competence.

28. according to the method for claim 27, wherein immunologic competence comprises the destruction of disease cell, infected cell and pathogenic agent.

29. according to the method for claim 27, wherein this compound exists with a certain amount of, makes this compound per 10 ⁸Form about 1 to about 10 in the individual white corpuscle genomic dna base pair ⁴Individual adducts.

30. according to the method for claim 27, wherein this compound exists with a certain amount of, makes this compound per 10 ⁸Form about 5 to about 10 in the individual white corpuscle genomic dna base pair ³Individual adducts.

31. according to the method for claim 27, wherein unreacted compound is removed.

32., wherein remove unreacted compound by in reaction mixture, adding quencher according to the method for claim 31.

33. cell colony according to the method preparation of claim 27.

34. a cell colony, its character is equivalent to the colony according to claim 33.

35. a cell colony, its character is equivalent to the colony according to claim 1.

36., breed at least 90% the T cell in the wherein processed leukocyte population and be suppressed according to the method for claim 27.

37. according to the method for claim 27, wherein this compound contain a kind of can with the non-covalent bonded nucleic acid binding moiety of nucleic acid.

38. according to the method for claim 27, wherein this compound contains:

(a) a kind of nucleic acid binding moiety;

(b) a kind of effector part that can form covalent linkage with nucleic acid; With

(c) the fragility linker of a kind of covalently bound nucleic acid binding moiety and effector part.

39. the method for claim 38, wherein nucleic acid binding moiety is a kind of aromatics insert, and effector partly is a kind of mustard seed base.

40. according to the method for claim 39, wherein this compound is to have general formula:

Two (2-chloroethyl) amino of N-(acridine-9-yl) Beta-alanine 2-[] ethyl ester (S-303) and salt thereof.

41. the method for claim 27, wherein this compound also is a kind of replication inhibitors.

42. the method for claim 27, wherein this compound also is a kind of topoisomerase enzyme inhibitor.

43. the method for claim 42, wherein this compound is selected from camptothecine and daunomycin.

44. the method for claim 27, but wherein this compound contains a kind of photoactivation part, and it forms covalent linkage with nucleic acid behind electromagnetic stimulation.

45. the method for claim 44, wherein this method further comprises reaction mixture is exposed to light, but photoactivation should photoactivation part, thereby but causes the formation of covalent linkage between photoactivation part and white corpuscle genomic dna.

46. the method for claim 45, wherein this compound is selected from: furocoumarin(e), actinomycin, anthracene nucleus ketone, anthramycin, benzodipyrane ketone, fluorenes, Fluorenone, single star fat indigo plant, norphillin A, organic dye; Phenanthridines, thiodiphenylamine sulfosalt, azophenlyene, thiodiphenylamine, triazobenzene, quinoline and thioxanthone, acridine and ellipticine.

47. the method for claim 46, wherein furocoumarin(e) is a kind of psoralene.

48. the method for claim 47, wherein psoralene is selected from: PAP, 8-methoxypsoralen (8-MOP), 4 '-amino methyl-4,5 ', 8-trimethylpsoralen (AMT), 5-MOP (5-MOP) and trioxa quinoline 4,5 ', the 8-trimethylpsoralen.

49. the method for claim 47 wherein makes this reaction mixture be exposed under the ultraviolet ray that wavelength is 200-450nm.

50. the method for claim 49 wherein makes this reaction mixture be exposed under the ultraviolet ray that wavelength is 320-400nm.

51. the method for claim 50, wherein making this reaction mixture be exposed to dosage is 10 ^-3-100J/cm ²Ultraviolet ray under.

52. the method for claim 51 wherein makes this reaction mixture expose for some time of 1 second-60 minutes under ultraviolet ray.

53. the method for claim 52, wherein psoralene is with 10 ^-4The concentration of-150 μ M exists.

54. the method for claim 53, wherein this psoralene is for having general formula:

4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen (S-59) and salt thereof.

55. the method for claim 54, wherein the concentration of S-59 is 10 ^-3-150 μ M.

56. the method for claim 55 wherein makes this reaction mixture be exposed under the ultraviolet ray that wavelength is 200-450nm.

57. the method for claim 56 wherein makes this reaction mixture be exposed under the ultraviolet ray that wavelength is 320-400nm.

58. the method for claim 57, wherein making this reaction mixture be exposed to dosage is 10 ^-3-100J/cm ²Ultraviolet ray under.

59. the method for claim 58, wherein making this reaction mixture be exposed to dosage is 3J/cm ²Ultraviolet ray under.

60. the method for claim 58 wherein makes this reaction mixture expose for some time of 1 second-60 minutes under ultraviolet ray.

61. the method for claim 60 wherein makes this reaction mixture expose about 1 minute time under ultraviolet ray.

62. the method for claim 61, wherein the cell density of leukocyte population is every milliliter of 10-10 ⁹Individual cell.

63. the method for claim 62, wherein the cell density of leukocyte population is every milliliter 2 * 10 ⁶Individual cell.

64. cell colony that produces according to the method for claim 38.

65. cell colony that produces according to the method for claim 40.

66. cell colony that produces according to the method for claim 41.

67. cell colony that produces according to the method for claim 46.

68. cell colony that produces according to the method for claim 54.

69. cell colony that produces according to the method for claim 59.

70. cell colony that produces according to the method for claim 63.

71. one kind promotes disease cell or pathogenic agent destructive method, comprises the leukocyte population of claim 1 is mixed mutually with the homogeneous variant cell colony of containing disease cell or pathogenic agent.

72. one kind promotes disease cell or pathogenic agent destructive method, comprises the leukocyte population of claim 70 is mixed mutually with the homogeneous variant cell colony of containing disease cell or pathogenic agent.

73. the method for claim 72, wherein this disease cell is a kind of cancer cells.

74. the method for claim 73, wherein this cancer cells is selected from: chronic lymphocytic leukemia (CML) cell, chronic myelomonocytic leukemia (CmML) cell, lymphocytic leukemia (CLL) cell, acute myelogenous leukemia (AML) cell, acute lymphoblastic leukemia (ALL) cell, multiple myeloma (MM) cell, He Jiejin lymphomas cell and non_hodgkin lymphoma cell.

75. the method for claim 74, wherein cancer cells is a chronic myeloid leukemia cells.

76. the method for claim 74, wherein cancer cells is a multiple myeloma cells.

77. the method for claim 73, wherein cancer cells is selected from: breast cancer cell, lung carcinoma cell, ovarian cancer cell, testicular cancer cell, prostate cancer cell, colon cancer cell, melanoma cells, kidney cancer cell, neuroblast oncocyte, a cancer cells and neck cancer cells.

78. the method for claim 71, wherein the disease cell is an infected cell.

79. the method for claim 78, wherein infected cell is by virus infection.

80. the method for claim 79, wherein this virus is selected from: cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) and kaposi sarcoma-associate herpesvirus.

81. the method for claim 71 wherein makes leukocyte population mix mutually in body with the homogeneous variant cell colony of mammalian hosts by the donor white corpuscle is imported among the described host.

82. the method for claim 81, wherein this mammalian hosts suffers from the recurrence of leukemia after the bone marrow transplantation or multiple myeloma.

83. the method for claim 71, its moderate stimulation leukocyte population is with the quantity of expansion to the special cytotoxic T cell of disease cell or pathogen antigen.

84. the method for claim 83 wherein stimulated separate leukocyte population from donor before in vivo.

85. the method for claim 84 is wherein by stimulating with the inoculation of antigen dialogue cell donor.

86. the method for claim 85, wherein the disease cell is the CML cell, and with the bcr-abl antigen inoculation white corpuscle donor of CML cell.

87. the method for claim 85, wherein the disease cell is a multiple myeloma cells, and inoculates the white corpuscle donor with myeloma cell's idiotype antigen.

88. the method for claim 83 stimulates wherein strippedly.

89. the method for claim 83, its moderate stimulation result from leukocyte population and contact with disease is intercellular.

90. the method for claim 83, its moderate stimulation result from contacting between leukocyte population and the antigen presenting cell, wherein this antigen presenting cell has contacted the disease cell.

91. the method for claim 83, its moderate stimulation result from contacting between leukocyte population and the antigen presenting cell, wherein this antigen presenting cell has contacted a kind of antigen of disease cell.

92. a method for preparing according to the cell colony of claim 1, wherein this method comprises:

(a) form a kind of vitro reactions mixture that contains leukocyte population and duplicate the inhibition compound; With

(b) this reaction mixture of incubation under certain condition, this condition can produce a kind of cell colony that contains leukocyte population, wherein the part of this leukocyte population is not bred, make that inducing of graft versus host disease (GVHD) is suppressed in the acceptor, and the part of this leukocyte population keeps immunologic competence.

93. the method for claim 92, wherein this compound is a kind of topoisomerase enzyme inhibitor.

94. the method for claim 93, wherein this compound is selected from camptothecine and daunomycin.

95. a method for preparing treated leukocyte population, wherein this leukocyte population is not bred generally, and can not cause graft versus host disease (GVHD) in the allogeneic host, and it comprises the following steps:

I) provide the sample of the leukocyte population of purifying; With

Ii) make the white corpuscle sample and can combine with certain amount, make this compound in per 108 white corpuscle genomic dna base pairs, form about 1-10 with the compound that nucleic acid forms covalent linkage ⁴Individual adducts, thus suppress propagation but keep leukocyte population to promote disease cell or pathogenic agent destructive validity.

96. a method for preparing the irritation cell that is used for mixed lymphocyte reacion is wherein handled the cell colony that contains leukocyte population according to the method for claim 27, and with treated cell colony as irritation cell.

97. whether definite propagation is the necessary method of cell function, comprises the following steps:

(a) form a kind of vitro reactions mixture, it contains a kind of cell colony and a kind of compound that can form covalent linkage with nucleic acid;

(b) this reaction mixture of incubation under certain condition, this condition can produce a kind of can not DNA synthetic but can RNA and the biosynthetic cell colony of protein; And

Observation of cell colony determines whether cell function exercises.

98. the method for claim 97, wherein this cell function is differentiation.

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