JP2003520563A - Methods for treating leukocytes, leukocyte compositions and methods of using the same - Google Patents

Abstract

  (57) [Summary] The present invention treats leukocytes to stop leukocyte proliferation and renders those cells ineffective in inducing graft versus host disease (GVHD), but to enhance the transplantation of allogeneic donor cells. Methods and compositions that are effective and promote the destruction of diseased cells or virulence factors are provided. Also provided are leukocyte compositions and methods of using these compositions to facilitate various types of immune reconstitution and immunotherapy to alleviate disease and to enhance the transplantation of allogeneic donor cells. You.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Patent Application No. 60 / 053,599 (filed Jul. 21, 1997).

FIELD OF THE INVENTION The present invention relates to cell compositions that are non-proliferative, but retain function, and methods of preparing and using these compositions. More particularly, the present invention relates to methods for the preparation of growth-inhibited leukocytes for use in infusion.

BACKGROUND OF THE INVENTION Bone marrow transplantation (BMT) is associated with a variety of malignant and non-malignant hematopoietic disorders (leukemia, multiple myeloma, lymphoma, anemia and immunodeficiency disorders such as severe combined immunodeficiency (SCI).
D), including Viscott-Aldrich syndrome and aplastic anemia).

Leukemia is a malignant neoplasm of the hematopoietic tissue. These neoplasms fall into two main forms: chronic and acute. Acute leukemia is characterized by an undifferentiated cell population (eg, acute lymphocytic leukemia or “ALL” and acute myelogenous leukemia or “AML”), whereas chronic leukemia usually exhibits a more mature form (
For example, chronic myelogenous leukemia or "CML" and chronic lymphocytic leukemia or "
CLL "). In 1995, there were approximately 25,700 new cases of leukemia in the United States, of which about 4,500 were CML. In 1996,
Approximately 7,000 new cases of CML have been diagnosed in the United States, of which approximately 30% eventually undergo allogeneic BMT (Leukemia So.
ciency: Leukemia Society Web Site, 1997.
). Approximately 50% of BMT patients are predicted to develop leukemia recurrence.

The general goal of leukemia therapy is to stop the growth of abnormal morphologies and to restore “normal” hematopoiesis in the bone marrow. The treatment regimen may include a combination of chemotherapy, radiation therapy, and bone marrow transplant. In the "generic" allogeneic bone marrow transplant (allogeneic BMT) protocol, leukemia patients are first given a bone marrow ablation dose of chemotherapy. At this stage of treatment, it is designed to eradicate all leukemia cells and their progeny. Discontinuation of pathological hematopoietic physiology subsequently replaces the functionally inoperable bone marrow with allogeneic disease-free stem cells. In an optimal setting, this transplanted bone marrow facilitates the restoration of normal polyclonal hematopoietic growth. The improvement in disease control also depends on the immune-mediated response of the graft to leukemic cells, which is called the anti-leukemic graft (GVL) effect.

Bone marrow is not the exclusive source of hematopoietic progeny. Peripheral blood and cord blood are also
It can be used as a source of stem cells. McCullough, The New
Generation of Blood Components, Trans
fusion, 35: 374 (1995). A trophic factor, such as a granulocyte colony stimulating factor, can be administered systemically to increase peripheral blood stem cell proliferation, thereby making a bone marrow replacement for the collection of hematopoietic progeny.

Unfortunately, the main barrier to successful BMT is the graft-versus-host disease (G
VHD), infection and recurrence of latent disease. GVHD and infection account for 10-30% of mortality and mortality during the first 100 days after transplantation (Basic).
and Clinical Immunology, 8th Edition, Daniel P
． States, Abb. Terr and Tristrom G.M. P
arslow (ed.) Appleton & Lange, Norwalk, CT
, 1994). If BMT fails, "recurrence" can result from the undetected growth of residual tumor cells. Slavin et al., "Immunotherapy of
Minimal Residual Disease by Immunoc
independent Lymphocytes and their Activ
ation Cytokines ", Cancer Investigatio
n, 10: 221 (1992). Medical treatment options in the past have been very limited under these circumstances. For example, long term use of a second bone marrow transplant and / or a cytotoxic drug is associated with poor survival.

Nevertheless, treatment strategies have emerged from a more complete understanding of transplant biology. Studies on BMT with or without T cell depletion indicate that T cells are important for anti-leukemic treatment, which is useful in CML patients in the management of minimal disease residuals or in the "complete blow" of relapse. Can be. With this understanding, treatment of leukemia patients with donor leukocyte infusion (DLI) after bone marrow transplantation has become an accepted treatment. Kolb et al., Blood 76: 2462 (1990);
Lim, S.L. H. Et al., Am. J. Hem. 54: 61-67, 1997; Gir.
Alt, S.S. A. Cur. Op. Onc. 8: 96-102, 1996; B.
arrett, J .; Cur. Op. Onc. 8: 89-95.
Leukocyte infusion was also used as an adoptive immunotherapy after bone marrow transplant failure to induce homeostatic remission. J. McCullough, "The New Gen
"Translation of Blood Components," Transfu
sion 35: 374 (1995). DLI is particularly effective in treating CML, but is also used for treating AML, ALL and CLL. DLI has also been used for the treatment of myelodysplastic (MDS), non-Hodgkin's lymphoma, Hodgkin's lymphoma and multiple myeloma. The infused cells appear to generate an immune response against the recipient's malignant cells. Approximately 70% of patients with CML relapsing into the chronic phase achieved recovery of persistent cell development after DLI. And it was negative for residual disease by PCR analysis for bcr-abl rearrangement. (Philadelphia chromosome) (Dr
obyski, W.W. R. Et al., Blood 82: 2310-2318, 1993.
).

However, this approach has risks, because GVHD is 80
%, And bone marrow anemia occurs in 56% of patients. These are the two major causes of treatment failure and the cause of death in up to 20% of responding patients (Champlin, R., et al., Act. Haemat. 95: 157-.
163, 1996). GVDH originates from donor T lymphocytes present in transfused BMT or DLI, proliferates and parasites in host tissues. Once expanded, donor T cells attack host tissues that produce pathological symptoms.

The eradication of disease after DLI is thought to be due to the immune response of tumor cell-grafts (ie the GVL effect) (Barrett, J. et al., Cur. Op. On).
c. 8: 89-95, 1996). The mechanism for the GVL effect is poorly understood. This is brought about by T cells from the donor. This is because T cell depleted BMT leads to a higher frequency of leukemia recurrence. An immune response is necessary to produce this effect, which was experimentally demonstrated by the higher rate of recurrence after allogeneic BMT compared to allogeneic BMT (Duell,
T. Ann. Int. Med. 126: 184-192, 1997). In Japan, a highly syngeneic population renders DLI treatment ineffective. Because random donors and hosts are not allogeneic enough (Takahashi K.
． Lancet 343: 700-702, 1994).

Clinical results suggest that this GVL effect is separable from pathogenic GVHD. Patients treated with T cell depleted allogeneic BMT showed a higher recurrence frequency than patients treated with BMT and standard GVHD prophylaxis, even when controlled for the number of GVHD occurrences (Horowitz, MM et al., Blo
od 75: 555-562, 1990). Many cases were also reported in which remission from CML was observed without the occurrence of GVHD (Duell, T. Ann. Int.
Med 126: 184-192, 1992). GVHD in animal models
Of GVL from GVL is achievable (eg, Johnson, BD, et al.
Bone Marrow Transplant. 11: 329-336, 19
93; Glass, B .; Br. J. Hem. 93: 412-420, 199
6; Johnson, B.H. D. Et al., Blood 85: 3302-3312,1.
995).

To avoid acute GVHD, several techniques were used to remove donor T lymphocytes or mature lymphocytes present in BMT. However, currently there is no effective treatment of bone marrow to avoid GVHD altogether. Because in most cases the GVL effect is immunocompromised, leading to higher recurrence rates (Kan).
tarjian, H .; M. Et al., Blood 87: 3069-3081, 199.
6). Some experimental approaches in human and animal models have included depletion of T cell subsets, UVB irradiation (Greenfield, JI, et al., J. Sur.
． Res. 60: 137-141, 1996), or T cell depletion followed by re-addition of a defined number of T cells, titration of a defined number of T cells in the absence of GVHD (
Drobyski, W.C. R. Et al., Blood, 82: 2310-2318, 19
93) is included. In one strategy, donor T cells are transfected with the herpesvirus thymidine kinase (HSV-tk) gene, a "suicide gene" that sensitizes the drug ganciclovir. If symptoms of GVHD are observed, the patient is given ganciclovir to clear T cells in vivo until the symptoms disappear (Bordignon, C. et al., Hum. G).
en. Ther. 6: 813-819, 1995; Bonini C .; Et Sc
ience 276: 1719-1724, 1997). Another "suicide gene" approach is the FAS gene (which encodes an apoptotic signaling protein
Ariad Pharmaceuticals) is used. In this FAS system, dimerizing agents such as FK1012 are used to induce FAS production, which kills donor cells expressing the gene.

While this “suicide gene” approach is perhaps attractive from the perspective of eliminating unwanted T cells after residual cancer has been treated, it has a number of significant drawbacks. First, the symptoms of GVHD are used as cues to initiate treatment with the drug. Therefore, GVHD must be started and then stopped. Second, the drug used to clear T cells must be administered systemically, which exposes the whole body to the drug for the time required to bring GVHD under control. Ganciclovir has toxic side effects and the HSV-tk protein is immunogenic. Moreover, this patient needs to be protected from the severe form of GVHD, so immunosuppressive prophylaxis remains an absolute requirement. Also, in some cases of chronic GVHD, the administration of ganciclovir proved to be completely ineffective (Bonini, C., et al., Science 276: 1719-1724, 1).
997). This is probably due to the discontinuation of this approach, where only proliferating cells are ganciclovir sensitive. Furthermore, even proliferating cells can be evaded from the effects of ganciclovir through the assistance of enzymes supplied by neighboring normal cells that are in the vicinity of HSV-tk cells (Good Samaritan effect).

In the FAS system, its drawbacks include the difficulty and insolubility of the dimerization agent that induces FAS production. The binding of the dimerizing agent to the FKBP protein in its host can limit the available drugs.

Moreover, the technical and practical problems associated with gene therapy make the application of this technology difficult to implement and control. The production of transfected cells is
There is a problem of low efficiency, which is due to the homogeneity of the treated cell population and hence GVHD.
It poses a problem with the ability to kill the cells completely when symptoms appear. The cells have to grow for one week, which means that there is a delay between when the cells are harvested and when the transfected cells are ready for injection. Finally, and most importantly, the cells injected into humans
It contains modified genetic material, which can prove to have deleterious effects on its host in the long run.

Ionizing radiation (γ irradiation or X-ray irradiation) or UV light (UVC λ = 200 to
280 nm; UVB λ = 280-320 nm; UVA λ = 320-400 n
m) is used to treat blood products to induce DNA damage in cells. Currently accepted methods for infusion related to GVHD prophylaxis include gamma irradiation treatment (25
00cGy). The clinical dose of gamma irradiation (2500 cGy) used for the prevention of TA-GVHD results in a 10 ⁵ to 10 ⁶ fold reduction of viable T cells. However, gamma irradiation and ionizing radiation are generally not specific for nucleic acids.
As a result of their high energy, gamma irradiation and UVC also attack proteins and other cellular components, resulting in significant non-specific damage (Deeg, HJ.
Et al., Blood Cells 18: 151-162, 1992). To prevent GVHD, UVB has been used to eliminate rat lymphocyte proliferation while preserving the survival of BM precursor cells and primitive hematopoietic stem cells for transplantation. 4,000
At a UVB treatment dose of 0 J / cm ² , CTL activity was reduced but still present (Gowing, H. et al. Blood 87: 1635-1643, 1996).
. However, UVB irradiation also leads to significant cell damage, especially when using lamps with a broad emission spectrum (Gowing, H. et al. Blood 87).
1635-1643, 1996; Pamphilon, A .; A. Blooo
d 77; 2072-2078, 1991). Surface molecular chemical alterations and membrane disruptions have been reported that lead to reduced cell viability and elicitation of immune responses, which presumably causes UVB to induce chemical changes to proteins and lipids, especially unsaturated bonds. Due to ability. These limitations indicate that UVB is not selective only for nucleic acids,
Due to the fact that it modifies any chemical groups that absorb between 280 and 320 nm.

The photosensitizer 8-methoxypsoralen (8-, used in the treatment of human leukocytes.
UVA irradiation in the presence of (MOP) was shown to inhibit both the stimulatory capacity and proliferation of peripheral blood leukocytes in mixed lymphocyte culture reactions in vitro (
Kraemer, K .; H. Et al., J. Inv. Derm. 77: 235-239,
1981; Kraemer, K .; H. Et al., J. Inv. Derm. 76: 80-
87, 1981). However, treatment of UVA and psoralens resulted in surface antigen expression, cytokine synthesis (IL-1, IL-6, as described in these reports.
, IL-8 and TNF) and inhibition of transcription of cytokine mRNA (Gruner, S. et al., Tiss. Ant. 27: 147-154, 198).
6; Neuner, P .; Et al., Photochem. Photobiol. 59:
182-188, 1994). Therefore, the photochemical treatment conditions described in these previous studies also inhibited T while inhibiting proliferation and reducing non-specific cell damage.
It inactivates cellular immune function; these treatment conditions do not appear to produce effective cells to provide some immune protection or induce GVL effects for the purpose of infusion. Finally, treatment of splenocytes and bone marrow cells with UVA and 8-MOP has been successfully used for reduction of GVHD in a mouse model (
Ullrich, S .; E. J. Inv. Derm. 96: 303-308, 1
991). As with all these previous reports, this study only examined GVHD in the context of donor leukocytes and did not provide leukocytes with immune protection or GVL function.

Clearly, there is a need for new approaches to suppress the induction of GVHD across allogeneic histocompatibility barriers that do not require systemic exposure to drugs. Such methods should provide some immune protection to immunocompromised individuals and / or kill residual cancer, but should prevent the growth of injected cells in their host.

The invention described and claimed herein provides for these and other problems associated with the prior art by providing a method of preparing leukocytes that do not induce GVHD but still maintain leukocyte function. Consider and overcome by. This and other advantages provided by the method of the present invention will be apparent from the detailed description below.

SUMMARY OF THE INVENTION The present invention treats leukocytes from an allogeneic donor and is unable to proliferate,
Provided is an effective method of producing leukocytes capable of maintaining survival and immune function effective in promoting the destruction of diseased cells or virulence factors. Since the treated leukocytes are unable to proliferate, they cannot induce GVHD when introduced into an allogeneic host (
GVHD is not a problem in syngeneic or autotransfusion), and is therefore ideal for use in DLI for the treatment of various cases and especially cancer, and to provide immune function to immunocompromised mammals. Is.

[0020]   Various aspects of the invention include the following.

[0021] An isolated cell population comprising a leukocyte population, wherein a portion of the leukocyte population is not proliferative, such that the cell population is associated with an allograft host allograft disease. (GVHD) cannot be induced; and a portion of this leukocyte population retains immunological activity, including the ability to promote destruction of diseased cells or virulence factors. Preferably, at least 90% of the white blood cells of this population are not proliferative.

The leukocyte population of the above embodiment is further characterized as: 1) preferably T cells, NK cells and antigen presenting cells; 2) upon proper stimulation, IL-2, IFN. -Ability to synthesize and / or secrete cytokines such as [gamma], IL-10 and GM-CSF; 3) inter alia CD4, CD8, CD
Capable of expressing surface markers characteristic of T cell activation and immune function such as 16 and CD56, and 4) exhibiting an increased ratio of killing to proliferative function relative to the untreated leukocyte population. thing.

The leukocyte population of the invention as a whole is unable to proliferate but retain immunological activity. This leukocyte population is capable of the following activities, among others:

[0024]   1. Accelerate the destruction of diseased cells, infected cells, and virulence factors.

2. Facilitating the seeding of a second population of cells. This second population of cells
It may be a suspension of cells or an organized collection of cells such as tissue cuts or organs. Such cells can include, for example, hematopoietic cells, myeloid cells, leukocytes, bone marrow cells, islet cells, hepatocytes, nerve cells, cardiomyocytes, stromal cells and endothelial cells.

[0026]   3. Promote immune reconstitution.

[0027]   4. Immunotherapy.

[0028]   5. Treatment of mixed chimerism.

[0029]   6. Promotion of the leukemia graft (GVL) response.

In one embodiment, the leukocyte population is effective in promoting the destruction of cancer cells. Preferably, the cancer cells are chronic myelogenous leukemia (CML) cells, chronic myelomonocytic leukemia (CMML) cells, chronic lymphocytic leukemia (CLL) cells, acute myelogenous leukemia (AML) cells, acute lymphoma leukemia (AML). ) Cells, multiple myeloma (MM) cells, Hodgkin lymphoma cells and non-Hodgkin lymphoma cells. In a preferred embodiment, the cancer cells are chronic myelogenous leukemia (CML) cells or multiple myeloma cells.

In another embodiment, the leukemia population is effective to promote the destruction of infected cells, diseased cells. Infected cells include cells infected with the virus. Preferably, the virus that infects the cell is cytomegalovirus (C
MV), Epstein-Barr virus (EBV), adenovirus (Ad), and Kaposi's sarcoma-associated herpesvirus.

[0032] In a further third embodiment, the leukemia population is effective to promote destruction of the virulence factor. Virulence factors include bacteria, fungi and parasites.

In yet another embodiment, the leukemia population is effective to facilitate implantation by a second population of cells. Cells used for implantation include, for example, hematopoietic cells, myeloid cells, leukocytes, bone marrow cells, islet cells, hepatocytes, nerve cells, cardiomyocytes,
It may include stromal cells and endothelial cells. Further, the second population of seeded cells may include solid organs or portions thereof.

Also provided by the invention is a subset of the leukocyte populations described above, selected by a variety of methods. This subset comprises lymphocytes and T lymphocytes, and the subset can be obtained, for example, by both positive and negative selection based on surface marker expression. Preferred surface markers are CD8, CD4,
Includes CD16 and CD56. Methods for selecting a subset of white blood cell populations from whole blood include white blood cell fractionation and red blood cell depletion.

In another embodiment, the invention provides various therapeutic methods utilizing the cell populations described above, which include donor leukocyte infusion, leukocyte add-back (add-ba).
ck), immune reconstitution, adoptive immunotherapy, treatment of mixed chimerism and methods for enhancing seeding of a second population of transplanted cells. For use in a method for enhancing the implantation of a second population of transplanted cells, a cell population of the invention is added to its host prior to, concurrently with, or after transplantation of the second population of cells. Can be introduced.

The invention also provides a method of preparing a treated leukocyte population, wherein the leukocyte population is generally non-proliferative and allograft disease in an allogeneic host. (VGDH) cannot be induced, and the method comprises the steps of: i) providing a sample containing a leukocyte population; and ii) a compound capable of forming a covalent bond with a nucleic acid, the compound comprising: Is a step of combining the leukocytes with an amount of about 1 to 10 ⁴ adducts per 10 ⁸ base pairs of genomic DNA, which inhibits proliferation, but immunological activity (the leukocyte population). (Including the ability to promote the destruction of diseased cells or virulence factors).

In a preferred embodiment, the compound is present in an amount effective to form an adduct of about 5-10 ³ per 10 ⁸ base pairs of leukocyte genomic DNA. Preferably, the method results in proliferation being inhibited in at least 90% of T cells in the treated leukocyte population.

[0038] Preferably, the compound capable of forming a covalent bond with a nucleic acid further comprises a nucleic acid binding moiety capable of non-covalently binding with a nucleic acid.

In one embodiment, the compound comprises: a nucleic acid binding moiety; a moiety capable of forming a covalent bond with a nucleic acid; and a frangible linker covalently linking the nucleic acid binding moiety and its effector moiety. Preferably the nucleic acid binding moiety is an aromatic intercalator and the effector moiety is a mustard.

In a preferred embodiment of the method for preparing a treated leukocyte population, the compound capable of forming a covalent bond with a nucleic acid comprises a photoactivatable moiety. This portion forms a covalent bond with a nucleic acid upon electromagnetic stimulation. When a compound having a photoactivatable moiety (ie, a photoactivatable compound) is used, the method further comprises exposing a sample of leukocytes combined with the compound to light to photoactivate the photosensitizing moiety, Thereby providing a photoactivated moiety that forms a covalent bond with leukocyte genomic DNA.

In one embodiment of the above method, the compound having a photoactivatable moiety is furocumarines, actinomycins, anthracyclinones, anthramycins, benzodipyrones, fluorenes, fluorenones, monostral. Lipid blue (monostral fats blue), norphyrin A, organic pigments, phenanthridines, phenazathionium salts, phenazines, phenothiazines, phenylazides, quinolines, thiaxanthones, acridines and ellipticenes Selected from the group. A preferred furocoumarin is psoralen. Preferred psoralens are PAP, 8-methoxypsoralen (8-MOP), 4
Includes'-aminomethyl 4,5 ', 8-trimethylpsoralen (AMT), 5-methoxypsoralen, and trioxalen 4,5', 8-trimethylpsoralen.

[0042]   If the compound used to treat the white blood cells is psoralen, this psoralen
Is preferably 10^-FourPresent in concentrations ranging from ~ 150 μM and of leukocytes
The sample has a wavelength in the range of 200-450 nm, preferably 320-400 nm.
Is exposed to UV light. Preferably, the ultraviolet light is 10^-3~ 100 J / cm ² Provided in a dose of. Leukocyte samples were exposed to UV light for a period of 1 second to 60 minutes.
Be exposed.

In a preferred embodiment, the method for preparing a leukocyte population treated according to the above embodiments uses psoralen and its salts, referred to herein as S-59. This psoralen has the following formula:

[0044]

[Chemical 3]

S-59 is in the range of 10 ^{−4 to} 150 μM, and even more preferably 10 ⁻³ to 15
Used at concentrations ranging from 0 μM. 20 leukocyte samples combined with S-59
Exposed to ultraviolet light having a wavelength in the range 0-450 nm, preferably 320-400 nm. The leukocyte sample combined with S-59 is preferably 10 ⁻³ -10.
It is exposed to UV light at a dose of 0 J / cm ² , more preferably 3 J / cm ² . In order to photoactivate this S-59, a leukocyte sample, preferably for 1 second to
Exposure to UV light for a period of 60 minutes, more preferably 1 minute. The leukocyte sample is preferably 10 to 10 ⁹ cells / mL, more preferably 10 ² to 10 ⁸ cells / mL.
Most preferably provided at a cell density of 2 × 10 ⁶ cells / mL.

Provided are leukocyte populations produced according to the above method, including leukocyte populations specifically treated with S-59.

Yet another aspect of the invention is a method of promoting the destruction of a diseased cell or virulence factor, which method comprises the leukocyte population produced by the above method and a homologous cell containing the diseased cell or virulence factor. Mixing with a population of allogeneic cells. This method can be performed in vitro or in vivo. In one preferred embodiment, the leukocyte population is mixed in vivo with its allogeneic cell population of a mammalian host by injecting donor leukocytes into the host. Preferably, the donor leukocyte infusion is administered to a mammalian host suffering from relapse of leukemia or multiple myeloma after MBT.

In a preferred embodiment of the method of promoting the destruction of diseased cells or virulence factors,
The diseased cells are cancer cells. Cancer cells from the following cancers are included: chronic myelogenous leukemia (CML) cells, chronic myelomonocytic leukemia (CMML) cells, chronic lymphocytic leukemia (CLL) cells, acute myelogenous leukemia (AML) cells, Acute lymphoma leukemia (ALL) cells, multiple myeloma (MM) cells, Hodgkin lymphoma cells and non-Hodgkin lymphoma cells. In the most preferred embodiment, the cancer cells are chronic myelogenous leukemia cells or multiple myeloma cells.

Another type of preferred cancer cells are breast cancer cells, lung cancer cells, ovarian cancer cells, testicular cancer cells, prostate cancer cells, colon cancer cells, melanoma cells, kidney cancer cells, neuroma cells, brain tumor cells and It is selected from the group consisting of cervical cancer cells.

In another preferred embodiment of the method of promoting the destruction of diseased cells or virulence factors, the diseased cells are infected cells. Preferred infected cells include cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (A
d), and cells infected with viruses such as Kaposi's sarcoma-associated herpesvirus.

In yet another preferred embodiment of the above method of promoting the destruction of a diseased cell or virulence factor, the leukocyte population is stimulated with one or more epitopes of an antigen specific for this diseased cell or virulence factor. , The number of cytotoxic T cells specific for that antigen has been increased. Stimulation or antigen-specific T cell expansion can be achieved in vivo,
It can be performed ex vivo or in vitro. In vivo stimulation is performed by vaccination of leukocyte donors with an antigen specific for this diseased cell or virulence factor prior to isolation of the leukocyte population from the donor. This disease is C
In the case of ML, the leukocyte population is preferably bcr-a of CML cells.
Stimulated with the bl antigen. In the case of multiple myeloma, the leukocyte donor may be vaccinated with the idiotypic antigen of the patient's myeloma cells.

In yet another embodiment of the above method of promoting the destruction of diseased cells or virulence factors, the leukocyte population is stimulated with mitogen. The stimulation is preferably performed in vitro with a mitotic composition such as phorbol myristate acetate (PMA) and ionomycin, or phytohemagglutinin.

Detailed Description Abbreviations The following abbreviations are used: BMT: bone marrow transplant DLI: donor leukocyte infusion (object) LDA: limiting dilution assay MLR: mixed lymphocyte reaction PA: photochemically stopped; PCT: Photochemical treatment; PI-DLI: injection of photochemically inactivated donor leukocytes (object); PUVA: psoralen and ultraviolet A irradiation. PAP: 4'and 5'primary amino substituted psoralen S-59: primary amino substituted psoralen having the formula:

[0054]

[Chemical 4]

PBSC: Peripheral Blood Stem Cell PC: Platelet Concentrate TA-GVHD: Transplantation-Associated Versus Host Graft Disease PHA: Phytohemagglutinin (Definition) “Allogeneic” is a non-genetically identical member of the same species. (I.e., its members are not related).

“Host” is used interchangeably with “recipient” and refers to a mammalian recipient of allogeneic donor leukocytes. Generally, the host is human, but other mammals (eg, mouse, dog, cat, monkey, horse, etc.) are also included.

“White blood cell” refers to any white blood cell, including lineage progenitor cells. "Antigen-presenting cells" include macrophages, and dendritic cells that are present in small numbers in peripheral blood, as well as their respective precursor cells. White blood cells are present in the circulating blood and bone marrow as well as in bone marrow hematopoietic sites, lymphoid sites and retinal sites of the reticuloendothelial system.

“Leukocyte population”, as used herein, refers to a leukocyte population that comprises more than one leukocyte type, and includes at least T cells, antigen presenting cells and NK cells.

“Donor leukocyte” refers to a leukocyte that is not endogenous to the host animal but is derived from an allogeneic donor. For in vitro and non-human applications, leukocyte populations and donor leukocytes can be derived from mammals, including rodents, rabbits, dogs and the like.

“Purified” means that the leukocyte population is isolated from the body and is substantially free of non-leukocyte cell types, and preferably such as cell debris present in the source of leukocytes. It is meant to be treated free of other contaminants, the source of which is also typically peripheral blood, bone marrow or splenocytes. Preferably, the leukocyte population is 1
Less than 3%, more preferably less than 5%, even more preferably less than 1% red blood cells (
RBC). In the most preferred embodiment, the hematocrit level is less than 0.5%. The method for purification of white blood cells is described below.

“Treatmented (treated) leukocyte” refers to a leukocyte that has been exposed to or contacted with a compound capable of forming one or more covalent bonds with a nucleic acid. This white blood cell is PCT
Can be "treated" by or with an alkylator-compound. "Photochemically inactivated" or "photochemically arrested" (PA) or "PCT arrested" leukocytes refers to PCT leukocytes that are unable to proliferate upon stimulation, and the leukocytes are proteins It is not meant to be inactivated in expression and immune function.

The leukocyte population of the present invention includes, but is not limited to, cells that are “non-proliferative”. A cell is "non-proliferative" if it has arrested DNA replication and is therefore unable to divide to produce new cells upon stimulation with mitogens, cytokines, antigens, antibodies or other growth stimuli. Say. It is not essential that the method of the invention result in the replication arrest of all leukocytes in the preparation. For the purposes of the present invention, the leukocyte population is at least 90%, more preferably at least 90% in the purified population if the few leukocytes capable of proliferation are insufficient to generate GVHD in an allogeneic host. It is appropriate that growth is stopped by 95%. In the most preferred embodiment, 99% or more of the white blood cells in this population are non-proliferative.
Proliferative activity can be assayed, for example, by limiting dilution assay (LDA) or separate ³ H thymidine incorporation, or as part of the MLR assay as described in the Examples below. In the most preferred embodiment, the T cells in the population are
Proliferation inhibited to the limit of detection by LDA. The results of these assays were shown to be an indicator of the ability of treated leukocytes to proliferate in vivo.

“Host graft disease” or “GVHD” refers to transplanted BMT or DLI
Resulting from clonal expansion, proliferation and parasitism of host cells of donor T cells present in The incidence and severity of GVHD has been shown to be associated with the presence of cloneable T cells (Kernan, NA et al. Blood 68: 770-7.
73, 1986). In patients who are HLA-compatible with their donor, the occurrence of GVHD is due to a minor histocompatibility antigen. The development of GVHD is due to the absence of immune function in immunosuppressed patients, a condition that requires BMT to be successful. Donor T cells can proliferate and challenge host cells in their environment unchallenged, which results in pathological conditions. At the cellular level, the antigen-presenting cells of the host are the major histocompatibility complex (
In the context of MHC) I and II, it interacts with its donor T cells and induces their activation towards cells with host-specific (non-major) antigens. This leads to clonal expansion of activated T cells, which attacks its host tissue and releases cytokines. The cytokines secreted by the T cells also activate various other effector cells of the host, which increase their tissue damage through the production of additional cytokines (cytokine storms). For example, Burakoff, S .; J. Graft-Versus-Hos
t Disease Immunology, Pathophysiology
, And Treatment, Brinkhouse KMS, S.M. A. (Edit)
: Hematology, Volume 12 (First Edition), New York, Marce
ll Dekker, Inc. , 1990, 725, Burakoff, S .;
J. Graft-Versus-Host Disease Immunol
Ogy, Pathophysiology, and Treatment, Br
inkhous KMS, S.M. A. (Edit): Hematology, Volume 12 (
1st Edition), New York, Marcell Dekker, Inc. , 19
90, 725.

When GVHD is diagnosed, the patient is usually given a high dose of an immunosuppressive drug that suppresses the GVHD. However, these drugs also render the graft leukocytes ineffective in GVL, and the patient can relapse with leukemia.

The ability of leukocyte populations to elicit GVHD is demonstrated in vivo, for example in Example 4 below.
Limiting dilution assay (as described in Example 5 below or in Example 5 below).
LDA) or by MLR in vitro. This leukocyte population had approximately 10 ⁻ the number of clonable T cells in that population that were present in the untreated leukocyte population control population (positive control for GVHD and proliferation) when assayed by LDA. ^When it is within the range of ³ to 10 ^-4 , it is judged that GVHD cannot be induced. In vivo, the lack of clinical manifestations of GVHD in allogeneic recipients of that leukocyte population (as described in Example 4) suggests that the population is incapable of causing GVHD.

A leukocyte population is either directly or indirectly associated with an immune response in which it is effective in killing or eliminating targeted diseased cells, pathogenic agents, or limiting the growth of the diseased cells or pathogenic agents from the body. It is considered "effective in promoting the destruction of diseased cells or virulence factors" if it contains leukocytes that may be involved in the disease. It is not necessary that all leukocytes present in the leukocyte population be able to promote destruction, but the population as a whole should be effective in this agar.

It should be appreciated that the diseased cell or virulence factor can be destroyed via any mechanism. It is not the intent of the present invention to have a method of treating leukocytes whose treated leukocytes are limited by a particular mechanism that can mediate destruction. The diseased cells can be destroyed by cytolysis by T or NK cells, but killed by other mechanisms such as by inducing them to undergo apoptosis. The diseased cell or virulence factor also has antibody-dependent cell-mediated cytotoxicity (A
DCC) can be destroyed by phagocytosis by macrophages, or eliminated by reticuloendothelial cell lines via any immune mechanism naturally present in mammals. A virulence factor present in a host cell can be indirectly destroyed by killing the host cell on which the virulence factor depends for its survival. The climate by which the immune system removes malignant cells, infected cells, or pathogenic agents from the body is well known in standard immunology textbooks, such as the Basics and Clinical Immun.
8th edition, Daniel P. et al. States, Abba I, Ter
r and Tristrom G.R. Parslow (eds.), Appleton and Lange, Norwalk, CT, 1994. Donor leukocytes that have been “effectively” treated have effector cells (eg, cytotoxic T cells (CT
L), NK cells, or macrophages, which directly kill the targeted diseased cell or pathogenic factor, or cells which induce the diseased cell to undergo apoptosis. Alternatively, the treated donor white blood cells may be replaced with other white blood cells (
Destruction may be mediated by stimulating killing or removal of white blood cells from the transplanted bone marrow, former DLI, or white blood cells of the host itself).
Stimulation of other white blood cells can be by surface antigen expression, cytokine secretion or any suitable mechanism. Cytolysis can occur via MHC-restricted and / or non-MHC-restricted (eg, by NK cells) mechanisms.

The efficiency of a leukocyte population in promoting the destruction of diseased cells or virulence factors can be measured by various assays, such as the MLR or ⁵¹ Cr release assay, as described in the Examples below. Cytolytic efficiency is demonstrated, for example, by the ability to mediate killing of leukemic cells in a ⁵¹ Cr release assay. For purposes of the present invention, a leukocyte population will have a cytolytic activity in a suitable assay (eg, a ⁵¹ Cr release assay) that is at least 2 greater than that of the negative control population.
When presented at levels above 0%, it is considered effective in promoting the destruction of diseased cells.
The types of diseased cells and virulence factors targeted for killing are provided below.

In general, the efficiency of promoting destruction is necessary to maintain the leukocyte population at a level of protein synthesis that allows the production and secretion of proteins, including signaling molecules such as cytokines. Preferably, on the plasma membrane, the receptor,
There is also expression of surface antigens, including adhesion and costimulatory molecules. Cell survival and membrane integrity are important for mediating GVL effects, and “cell survival” is defined herein as survival in the circulation (ie, in the bloodstream and other tissues). .

While some cytokine production may be inhibited, preferably cytokine production is at least 70%, more preferably 80%, and even more preferably 90% of the level prior to treatment with the compounds described above. Above, and most preferably 9
Maintained above 5%. Preferably, the leukocyte population comprises leukocytes capable of secreting at least interleukin-2 (IL-2) and interferon-γ (IFN-γ) under stimulation. These cytokines activate T cell activation and GVHD
/ Relevant because of their established role in GVL. Other related cytokines include granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-10 (IL-10). Cytokine secretion can be readily assayed, for example, by using a cytokine assay kit (eg, a kit from R & D Systems) as described in the Examples section below.

The surface antigens expressed on leukocytes in the purified leukocyte population, which are also referred to as surface markers, depend on the particular cell type. Preferably, this population comprises leukocytes expressing the following surface antigens involved in interactions associated with T cell and NK cell activation and known to be involved in immune function: CD2,
CD28, CTLA4, CD40 ligand (gp39), CD18, CD25,
CD69 (lymphocyte activation marker) and CD16 / CD56, antigen. Preferably, the leukocytes also contain the following other surface markers: MHC class I and class II, CD8, CD4, CD3 / TcR (T cell receptor), adhesion molecules (eg CD54 (ICAM-1), LFA. -1 and VAL-4), and other costimulatory molecules. Expression of surface antigens can be achieved by a variety of assays, such as staining cells with antibodies specific for a particular antigen and detecting labeled antibodies directly or indirectly by standard FAC-SCAN analysis. It can be detected and measured. Other methods known in the art include immunoprecipitation of surface antigens and immunoblots.

“Affected cells,” as used herein, refer to cancer cells, infected cells, or cells in any type of pathological condition. These can be in vivo or in vitro. Cancer cells or malignant cells can be of any type of cancer, any tissue or cell type origin. Such malignant cells or cancer cells include, but are not limited to, the following malignant diseases: chronic myelogenous leukemia (CM).
L), leukemia including chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), acute lymphoma leukemia (ALL), multiple myeloma (MM), non-Hodgkin's lymphoma and Hodgkin's disease (lymphoma); breast cancer, lung Solid tumors including cancer, ovarian cancer, testicular cancer, prostate cancer, colon cancer, melanoma, kidney cancer, neuromas, brain tumors and cervical cancer. Infected cells include cells infected by any of the following: viruses, bacteria, fungi, parasites, or other pathogenic microorganisms.

A “pathogenic agent” is defined as any agent that includes nucleic acid and can cause disease in humans, other mammals, or vertebrates. Examples of virulence factors are humans,
Bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas that cause disease in other mammals or vertebrates. The genetic material of the virulence factor can be DNA or RNA, and the genetic material can be present as single-stranded or double-stranded nucleic acid. The virulence factor may be external to the cell or intracellular, as exemplified by HIV in macrophages.

The terms “infusion” and “transfusion” of donor leukocytes are used interchangeably herein and refer to the same procedure.

“Light dose or dose” or “PCT dose” refers to the total light energy per unit area received by a sample of white blood cells received during PCT, as measured in units of J / cm ^2. . The light dose can be altered by changing the intensity of the light and the time of exposure of the light to the cell sample.

“Intensity” of light refers to the energy of light received per unit of the sample per second, as measured in units of W / cm ² .

A “photoactivatable moiety” is defined herein as a moiety that undergoes a chemical change in response to electromagnetic radiation. When the compound containing the photoactivatable moiety is photoactivated by electromagnetic radiation, the photoactivatable moiety forms a covalent bond with the nucleic acid to form a compound: nucleic acid complex (herein referred to as “additional compound”). Object)).

“Aromatic intercalator” refers to a compound or region thereof that has an aromatic ring structure and is capable of intercalating into nucleic acids. Aromatic intercalators include, but are not limited to, anthracene, acridine, naphthalene, naphthonic acid, and the like.

“Isolated cell population” as used herein includes a population of cells that resides outside the organism in which the cell normally resides.

“Immunological activity” as used herein refers to T cells, B cells,
Refers to a function expressed by cells of the immune system such as NK cells, antigen presenting cells (APCs), and other cells, such functions including cytokine synthesis and secretion, cell-mediated cytotoxicity, antibody Including but not limited to synthesis and secretion, processing and presentation of antigens and antigen fragments, and expression of surface markers characteristic of immune cells.

INCORPORATION BY REFERENCE References, including patents, published patent applications and other publications, cited in this application are hereby incorporated by reference.

Description of the Preferred Embodiments Various preferred aspects of the present invention are summarized below and are further described and illustrated in the detailed description and examples that follow.

The present invention provides a GVH with a minimal isolated leukocyte population while still retaining sufficient immune function that is effective to promote destruction of diseased cells or virulence factors.
Methods are provided for treating a population to render a portion of the population non-proliferative to the extent that causes D. By "minimal GVHD", the degree of GVHD is insufficient to cause mortality in mammals, or promotes the destruction of diseased cells or virulence factors, and / or mediates GVL effects. It is meant to be insufficient to extinguish the collective ability.

GVHD is clearly associated with clonal expansion of donor T cells, and in principle, any treatment that removes, inactivates, or kills leukocytes is effective against it. On the other hand, GVL is associated with the immune response of donor leukocytes and may be the result of cytolytic effects, antigen presentation, synthesis of specific cytokines, or a combination of the above. This requires that some of the functions of donor leukocytes are intact for the observed GVL effect. Inactivation of donor leukocyte function in an attempt to avoid GVHD also provides the immunocompromised host, or BMT host, with donor BM or leukocytes that are ineffective in providing the necessary immune function.

While the treatment may eliminate GVHD in an allogeneic host, but still provide immunoprotection and / or induce GVL effects, while retaining some level of leukocyte function. However, the proliferative capacity of white blood cells must be selectively eliminated. The present invention provides leukocyte populations having these properties.

The isolated leukocyte populations of the present invention provide various advantages over those previously described and used. One significant advantage is that eliminating the onset of GVHD allows for safe infusion of donor leukocytes into the patient. As a result, a host lacking the immune system or having a chimeric immune system may accept either treated donor leukocytes and / or higher doses of multiple DLI, thus enhancing the efficacy of treatment of the disease. obtain. Previously, the GVHD threat limited the number of white blood cells injected.

The non-proliferating leukocyte population is effective in providing the host with sufficient immune protection to combat cancer and infection. Such leukocytes are recurrent hematological malignancies (eg, chronic myelogenous, acute myelogenous, acute lympholytic, multiple myeloma, as part of, or as an alternative to, BMT treatment. And other leukemias and myelomas). The GVL effect from DLI is not only useful in eliminating minimal residual disease, but also to a higher degree, especially since the absence of leukocyte proliferation allows safe infusion of higher cell numbers. It can also be applied to the treatment of malignant cell loads. The treated leukocyte populations of the invention are also transplanted for the treatment of certain solid tumors that are susceptible to immunomodulatory therapy (eg, breast cancer and renal cell carcinoma) and in both compatible and incompatible xeno-BMT procedures. It is useful for preventing unilateral rejection. Various other clinical applications for which the treated leukocytes may be applied in treatment are described in detail below.

The method of treating leukocytes of the present invention comprises: White blood cells can be obtained from bone marrow, cord blood, or whole blood. The most convenient source of white blood cells is peripheral blood. Methods of isolating leukocytes from other sources are described in the scientific literature. Whole blood is purified,
It is then processed to enrich leukocytes, for example by red blood cell depletion or leukopheresis. The resulting purified leukocyte population is approximately 99% free of red blood cells, which are of different size and density compared to white blood cells and are easily removed by these processes.

The leukocyte population is then mixed with the compound and treated under conditions effective to arrest the proliferation of leukocytes without compromising cell viability and integrity. Unreacted compound can be removed or the compound can become inactive over time so that removal is not necessary. In a preferred embodiment, "compound" refers to a compound capable of forming a covalent bond with a nucleic acid. The covalent bond formation may be formed from about 1 to 10 ⁴ adducts, preferably 5 to 10 ³ adducts, most preferably 10 ³ adducts per 10 ⁸ base pairs of leukocyte genomic DNA. It is important that the treatment conditions minimize nonspecific damage to proteins and other cellular components and avoid membrane damage. The membrane integrity of the majority of treated leukocytes is required for the GVL effect to occur. These treatment conditions also either make it impossible to induce GVHD in the treated leukocytes when introduced into an allogeneic host or render the treated leukocytes non-responsive in an in vitro MLR assay. It is effective to Further, the treated leukocytes should maintain effective protein expression in achieving immune function, specifically in vivo or in vitro, to promote destruction of the affected cells or virulence factors. is there.

The properties of suitable compounds capable of forming covalent bonds with nucleic acids, preferably double-stranded DNA, are disclosed below. The compounds can include moieties that can form covalent bonds with nucleic acids, which moieties are photoactivatable or chemically reactive. The compound inhibits cell growth by forming covalent bonds with genomic DNA, thus interfering with the function of DNA polymerase and rendering leukocytes non-proliferative.

The number of covalent adducts that the compound forms with nucleic acids of leukocytes can be regulated such that the compound inhibits proliferation but is effective in promoting the destruction of diseased cells or virulence factors. Keep functioning. With an alkylator compound, the effect can be modulated by adjusting the concentration of the compound and the length of time that leukocytes are contacted with the compound before unbound compound is removed. The concentration required will depend on the properties of the particular compound, such as its solubility in solution and DNA binding constant. The compound is typically about 1 to 10 ⁴ adducts per 10 ⁸ base pairs of leukocyte genomic DNA, preferably about 5 to 10 ³ adducts, and even more preferably about 10 ² to 10 ³ adducts. Used in a concentration effective to produce an adduct of a covalently bound compound. Ideally, the conditions for treating the leukocyte population are genomic DNA
Approximately 10 ³ adducts are produced per 10 ⁸ base pairs. The lowest concentration of compound effective to achieve the leukocyte compositions of the present invention is the preferred concentration.

With photoactivatable compounds, the PCT effect can be modulated by adjusting the concentration of the compound, the wavelength of light, and the length of time of exposure to light. The conditions for PCT using psoralen and other photoactivatable compounds are described in detail below.

The treatment conditions are: about 1 to 10 ⁴ covalent compound adducts per 10 ⁸ base pairs of leukocyte genomic DNA, preferably about 5 to 10 ⁴ adducts, and even more preferably about 1.
Targets to produce 0 ² to 10 ³ adducts. Ideally, the treatment conditions are
About 10 ³ adducts are produced per 10 ⁸ base pairs of genomic DNA. D resulting from treatment
The number of adducts of NA-compounds can be measured, for example, by using radiolabeled DNA binding compounds, as described in the Examples below.

Another type of compound that can be used in the treatment of leukocytes is a small molecule that acts as an inhibitor of DNA replication. Such small molecule replication inhibitors are
As mentioned above, optionally a linker (fragile or non-fragile) and an effector may be included. Any step of DNA replication can serve as a target for inhibition, including formation of the origin recognition complex (ORC), recruitment of ORC to the origin, initiation of replication, elongation reaction, etc.). In addition, inhibitors of enzymes that promote the process of replication, such as helicase and topoisomerase, are useful. Examples of topoisomerase inhibitors include, for example, camptothecin.
in) and daunomycin.

To combine a sample of white blood cells with a compound, the compound of the invention may be introduced into a suspension of white blood cells (white blood cell sample) in several forms. The compound can be introduced as an aqueous solution in water, saline, a synthetic medium such as "Sterilete® 3.0" or in a solution in which leukocytes are suspended. Suitable solutions for leukocyte resuspension (infusion grade and non-infusion grade) are provided below. "Synthetic medium" is defined herein as an aqueous synthetic storage medium for blood or blood products. The compound may further be provided as a dry formulation with or without adjuvant. The cell suspension is then agitated to mix in the compound.

Leukocytes treated with a compound are resuspended in a physiologically equilibrium solution (eg plasma, synthetic medium, or a combination thereof). White blood cells may be provided in a volume of 200 mL to 1 liter. The leukocytes for treatment are preferably contained in a reaction vessel such as a blood bag. Blood bags are known in the art.

The effect of compound treatment on leukocyte population viability and function is dependent on proliferation and G
It can be monitored by in vitro as well as in vivo assays to determine optimal treatment conditions that minimize VHD activity while maximizing cytotoxic function and / or GVL effects. Optimum conditions will vary with the nature of the compound used and will be tested for disease or virulence factors. In the case of photoactivatable compounds, the most preferred PCT conditions include the lowest concentration of the compound and the lowest light dose found to be sufficient to provide a leukocyte population in which growth is arrested, and the affected cells or pathogens. It is effective in promoting the destruction of factors. The treated leukocyte population is characterized as follows.

Cell viability of the treated leukocyte population is assessed. Cell viability is, for example,
It can be measured in vivo by PCR analysis to detect sequences specific to donor leukocytes as described in the Examples section below. Preferably, the leukocyte population has a cell viability of at least 3 weeks. The rapid clearance of leukocytes upon treatment can affect their ability to induce an anti-leukemic response. The membrane integrity of treated leukocytes is also required for the GVL effect to develop. Membrane integrity is
It can be determined by trypan blue or propidium iodide dye exclusion. PC
The T procedure, when used, maximizes the number of white blood cells with intact membranes,
Optimized for light doses and / or compound concentrations.

Proliferative activity can be measured, for example, by limiting dilution assay (as described in the Examples below).
LDA) or mixed lymphocyte reaction (MLR) assay. Leukocyte activity in the mixed lymphocyte reaction (MLR) assay was demonstrated by GVH in vivo.
Predict the ability of treated leukocytes to mediate D. Alternatively, GVHD can be measured by monitoring GVHD-induced GVHD symptoms and / or morbidity and mortality in MHC-matched animals infused with treated leukocytes.

Leukocyte activity can be determined by monitoring cytokine synthesis, expression of surface antigenic markers, and the ability to lyse target cells. Expression of surface antigenic markers is determined through the use of appropriate antigen-specific antibodies and standard FACS-SCAN analysis. Antigenic marker, CD2, CD28, CTLA4,
The presence of CD40 ligand (gp39), CD18, CD25, CD69 (a lymphocyte activation marker) and CD16 / CD56 (these are known to be associated with interactions related to T cell and NK cell activation and immune function). Has been)
Is determined as a function of covalent compound and PCT light dose concentration, if used. Given the mild nature of PCT on the protein,
The stability of surface molecules under PCT conditions is not expected to be affected. Treatment conditions, such as PCT, are selected such that they do not adversely affect the expression of surface molecules or reduce cytokine production below desired levels (preferred levels are disclosed above).

Cytolytic activity indicative of GVL activity can be measured, for example, by lysis of human or mouse leukemia cell lines in a ⁵¹ Cr release assay. The anti-leukemic effect may be assayed in vitro as described by Choudhury et al., Blood 89: 1133-1142, 1997, or in vivo as described by Johnson et al., Blood 85: 3302-3312, 1995. Can be assayed. For example, a mouse model of a solid tumor can be used to test activity against solid tumors. The ability to mediate lysis of infected cells or virulence factors can be assayed using infected animals in animal models. GVHD
And GVL capacity can also be measured in vivo as described in Examples 4 and 5 below.

All of the above assays are described in the examples below. Due to the similarities in immune responses between mouse and human, experiments conducted using the mouse model are predictive of human response. For example, the GVL effect has been well documented in both mouse and human systems. The results of these assays are predictive of in vivo biological response in DLI treated hosts.

The concentration of the compound determined to be effective from the results of the tests in the animal model is tested in a representative number of patients, and the concentration of the most effective compound that results in the alleviation of the disease in the patient is used. Most preferred are conditions that affect the majority of patients. The patient is generally a BM transplant patient.

From the results of such assays and tests, determining optimal treatment conditions that produce leukocyte populations that are incapable of proliferation and GVHD but are effective in promoting the destruction of diseased cells or virulence factors. It will be possible. The treated white blood cell population is G
It should have VL activity.

Leukocyte Donor Selection For DLI, the donor must be allogeneic with respect to the host of leukocyte infusion (recipient). HLA-A and -B are HLA I genes, and HLA-DR is HLA II gene. Each of these genes exists in several different allelic forms. Since each individual inherits two copies of chromosome 6 (including the HLA gene), individuals can have up to 6 different HLA-A,-
B, and -DR proteins (the products of two different alleles at each locus) can be expressed. Preferably, the allogeneic donor is genotyped at 3 or more of the 6 HLA loci of HLA-A, B, and DR. In a haploidal transplant, the donor and host are matched at 3 out of 6 HLA loci. Ideally, the donor and recipient are the same for HLA-A, B and DRBI. Unrelated, HLA matched donors can be searched through the US National Bone Marrow Donor Program (NMPD).

Currently, for patients up to 55 years of age who receive unmodified non-T cell depleted transplants,
Patient and donor HLA-A, B, and DRB1 genes must be identical. Patients aged 36 or younger have HLA-A, -B or -D
It can be transplanted from different donors by one or less minor antigen mismatches for R. A minor mismatch of HLA-A or B is defined as two antigens belonging to the same cross-reactive group. The minor nonconformity of HLA-DR is the same D
Defined as two haplotypes that express R-specificity but differ for the DRBI allele. Criteria for appropriate allogeneic donors are O'Re
illy, R.I. Et al., Allogeneic Marrow Transplan
ts: Approaches For The Patient Lackin
g A Donor, in Hematology-1996, The Edu
Cation Program of the American Society
See ty of Hematology, 132-146.

Leukocyte Preparation The starting materials for the leukocyte populations of the invention are, for example, regional blood, similar to those currently existing for processing peripheral blood stem cells as well as other sources of hematopoietic stem cells (eg, bone marrow). It can be provided by a process service center. Leukocytes from the donor can be apheresed (apheresis can be performed in various facilities, including regional blood centers or hospitals (blood banks)), and can be treated in any suitable facility equipped to perform the procedure. . Alternatively, apheresed leukocytes can be shipped overnight to a Good Manufacturing Practice (GMP) cell processing facility for treatment and quality assurance testing. The leukocytes treated for DLI can then be sent to the patient's hospital for administration to the patient by same day delivery. Alternatively, the treated leukocytes can be treated by methods known in the art, eg, at one of the rates of freezing.
Can be cryopreserved by control of 0% DMSO and storage in liquid nitrogen (R
ussel et al., Bone Marrow Transpl. 19: 861-86
6, 1997). If blood transfusion is required, the frozen white blood cells will be thawed and washed to remove DMSO.

One conventional method for the preparation of purified leukocytes involves isolation from whole blood by density gradient centrifugation. This typically involves isolation of T cell-rich white blood cells by centrifugation through a Ficoll gradient. For example, Longley and Stewart, J. et al. Immunol. Methods, 121, 33-3
8, 1989. This procedure is performed as follows: (1) freshly pumped blood sample (eg 50-200 ml) is carefully overlaid on Ficoll without interfering interface; (2) gradient Leukocytes are collected (after centrifugation) by removing the band of opaque cells located at the interface.
(3) The collected cells are washed to remove Ficoll. Red blood cells and granulocytes are
This system pelletizes. The cells are usually washed one or more times by centrifugation and supernatant removal to remove Ficoll. This procedure produces a purified leukocyte preparation, ie, a preparation that is substantially free of red blood cells.

There are automated methods for preparing large scale preparations of leukocytes. For example, the present invention provides a leukocyte apheresis machine (eg, COBE Spectra A
pheresis System, COBE BCT, Inc. Lakewoo
d, CO) is intended to process blood, as done according to the manufacturer's instructions. Preferably, an apheresis device that provides the leukocyte preparation with the lowest percent hematocrit is used. After leukapheresis, the leukocyte preparation may then be washed and diluted with an appropriate solution (plasma, infusion grade solution, etc., as described above) to achieve a final hematocrit of less than 0.5%. If the above procedure did not reduce hematocrit levels sufficiently,
Percoll separation can be applied as a further purification step. As an alternative to leukocyte apheresis, the red blood cell depletion method is well known to those skilled in the art.

A leukocyte population that comprises substantially only T cell and NK cell or other leukocyte combinations is desired, and the selected cell subset is, for example, positive using appropriate antibodies to identify specific cell surface markers. And / or use negative selection,
It can be isolated from leukocyte preparations by using the Fluorescent Cell Analysis Separation Method (FACS). Examples of surface markers that can be used for selection are CD4, CD8, CD16
And CD56, but is not limited thereto. Such a cell separation procedure
Well known in the art, for example Ed Harlow and David.
Lane, Antibodies A laboratory Manual,
Cold Spring Harbor Laboratory, Cold S
See pring Harbour, New York, 1988.

The purified leukocyte population is suspended in a suitable solution as disclosed above. The purified leukocyte population can be treated immediately or cryopreserved for later treatment. In preparation for growth inactivation, the purified, mixed leukocyte preparation is resuspended in an isotonic solution (eg, plasma, synthetic medium, or a combination of the two). The purified leukocyte population is generally 10 to 10 ⁹ per ml.
Cells are prepared at a cell density of preferably 10 ⁴ to 10 ⁷ cells / ml, ideally 2 × 10 ⁶ cells / ml.

The present invention contemplates various scenarios for preparation and administration or use of treated leukocytes. Peripheral blood (or an alternative source of white blood cells, including bone marrow or cord blood) is collected and frozen until ready for proliferative inactive treatment, at which point cells are thawed and cryopreserved. Washed to remove, optionally processed to obtain a purified leukocyte preparation, and subjected to treatment. The treated leukocyte population can then be used to infuse the patient. The following alternative scenarios are also possible: i) peripheral blood collection, processing,
Leukocyte treatment and infusion; ii) collection, processing, leukocyte treatment, freezing of treated cells, washout of cryopreservative, and infusion; iii) to donors with target antigens to expand antigen-specific T cells. Vaccination (one or more times), collection, processing, treatment and infusion of peripheral blood from vaccinated donors; iv
) In vitro sensitization of donor leukocytes with target antigens to expand antigen-specific T cells, leukocyte treatment, and infusion.

Compounds In one embodiment of the invention, compounds capable of forming covalent bonds with nucleic acids are suitable for the purpose of achieving the leukocyte population of the invention. These compounds preferably include moieties that bind non-covalently to nucleic acids, as well as the same or different moieties that can react to form covalent bonds with nucleic acids. The compound preferably has the following properties: i) high binding affinity for nucleic acids; ii) solubility in aqueous solution; and iii.
) Ability to penetrate the white blood cell membrane. Moieties that can form covalent bonds with nucleic acids are chemically reactive moieties that are activated and do not require external stimuli to react with nucleic acids, and only after stimulation in the form of electromagnetic radiation. A photoactivatable moiety that reacts with the nucleic acid is included.

As used herein, the term “alkylator compound” means at least one chemically capable of reacting with a nucleic acid to form a covalent bond in the absence of an external stimulus (eg, light stimulus). By a compound is meant a reactive moiety.

The compound capable of forming a covalent bond with a nucleic acid may further be photoactivatable.
A "photoactivatable compound" as defined herein includes moieties that are capable of forming covalent bonds with nucleic acids when photoactivated by stimulation with light of a particular wavelength.

Preferably, the compound comprises both a moiety capable of reacting with a nucleic acid to form a covalent bond and a moiety capable of non-covalently binding to a nucleic acid. Within the scope of the present invention, a moiety capable of binding a nucleic acid may also act as a moiety capable of reacting covalently with a nucleic acid and may be, for example, photoactivatable.

In one embodiment, the alkylator compound comprises: a nucleic acid binding moiety;
A moiety that is capable of reacting with a nucleic acid to form a covalent bond (referred to herein as an "effector molecule"); and a cleavable linker that covalently links the nucleic acid moiety and the effector moiety. In a preferred embodiment, at a suitable pH value in aqueous solution, these compounds have a period of activation during which they can bind and react with nucleic acids.
of activity). After this period of time, the compound decomposes into a product that no longer binds or reacts well with nucleic acids.

The chemical mechanism of these alkylator compounds can be broadly described as an anchor covalently attached to a cleavable linker, which covalently attaches to an effector. An “anchor” is also called “nucleic acid binding moiety”, and is a nucleic acid biopolymer (D
NA, RNA, or synthetic analogs thereof) are non-covalently bound moieties. An "effector" or "effector moiety" is defined as a moiety that reacts with a nucleic acid by the mechanism of forming a covalent bond with the nucleic acid. A "cleavable linker" is defined as a moiety that acts to covalently link an anchor to an effector and degrades under certain conditions so that the anchor and effector are no longer covalently linked. . The anchor-cleavable linker-effector arrangement allows the compound to bind specifically to nucleic acid (due to the binding ability of the anchor). This causes the effector to approach a reaction with nucleic acids.

Preferably, the nucleic acid binding moiety is an aromatic intercalator.
attor), acridine, acridine derivatives, ellipticene (elliptic)
enes), 2-polyamines, groove binders, and hydrophobic or form-selective binders. In a preferred embodiment, the cleavable linker is a forward ester group, a reverse ester group, a forward thioester group, a reverse thioester group, a forward and a reverse thionoester group, a forward and a reverse dithioacid group, a sulfate group, a forward and a reverse sulfonate group, a phosphate. Groups, and functional group units selected from the group consisting of forward and reverse phosphonate groups. The effector moiety preferably comprises a functional group unit selected from the group consisting of mustard groups, mustard group equivalents, epoxides, aldehydes, and formaldehyde synthons.

When an alkylator compound is combined with a leukocyte composition at physiological pH to form a reaction mixture, the effector moiety of the compound reacts with the nucleic acid present that it contacts. The effector moiety that does not react with nucleic acid is gradually hydrolyzed by the solvent. Hydrolysis of the cleavable linker coincides with effector-nucleic acid reaction and effector hydrolysis. It is desirable that the cleavable linker degrade at a rate slow enough to allow inactivation of leukocyte proliferation; that is, the rate of degradation of the cleavable linker is greater than the rate at which the compound reacts with leukocyte nucleic acid. slow. After a sufficient amount of time has passed, the compound will have an anchor (which may also have a fragment of the cleavable linker) and an effector-nucleolytic product (wherein the fragment of the cleavable linker also has an effector. Or an anchor (which may also have a fragment of the cleavable linker) and a hydrolyzed effector degradation product (wherein the fragment of the cleavable linker is also
Can remain attached to the effector). Additional fragments of cleavable linkers can also occur upon degradation of compounds that do not remain attached to either anchors or effectors. The exact embodiments of the alkylator compounds of the present invention include whether the anchor or effector degradation product has a fragment of the cleavable linker, or is attached to either the anchor or effector degradation product. Determine if additional fragments of the cleavable linker that do not remain are generated.

Preferred alkylator compounds are those which upon cleavage of the cleavable linker produce degradation products which are less mutagenic. The mutagenicity of the compound is primarily due to the anchor moiety after effector degradation. This is because anchors can have the ability to interact with nucleic acids and interfere with nucleic acid replication even when the effector moieties are hydrolyzed. After the cleavable linker is cleaved, the anchor fragment has a substantially reduced mutagenicity.

In a further embodiment of the invention, compounds capable of forming covalent bonds with nucleic acids are removed from the reaction mixture after a certain period of time. The optimal reaction time can be determined empirically, as described below in the examples. Procedures and compositions for removing compounds from reaction mixtures are provided below: co-owned US patent application Ser. Nos. 08 / 779,830; 08 / 779,885; and ibid. 09 / 003,113; and co-owned U.S. patent application attorney docket no. 28
217-2000.21 and 28217-2000.22 (both filed on July 8, 1998). The entire disclosures of the patent applications mentioned in the preceding sentence are incorporated herein by reference in their entirety. Alternatively, a quenching agent that reacts with and inactivates the compound can be added to the reaction mixture. Exemplary quenching agents and methods are disclosed in commonly owned US Provisional Patent Application No. 60/0.
70,597 (filed Jan. 6, 1998) and U.S. Patent Application Attorney Docket No. 28217-20006.00 (filed Jul. 6, 1998). All disclosures are incorporated herein by reference in their entireties.

A wide variety of groups are useful for use as anchors, linkers, and effectors. Examples of anchor groups that can be used in the alkylator compound include:
Intercalators (including aromatic intercalators), minor groove binders, major groove binders, molecules that bind by electrostatic or hydrophobic interactions, and molecules that bind by sequence-specific interactions include, but are not limited to Not done. The following is a non-limiting list of possible anchor groups: acridine (and acridine derivatives (eg, proflavine, acriflavine, diacridine, acridone, benzacridine, quinacrine)), actinomycin, anthracyclinone, rhodomycin. , Daunomycin, thioxanthenone (and thioxanthenone derivatives (eg, miracil D)), anthramycin, mitomycin, etinomycin (quinomycin A), triostin (
triostin, ellipticine (and its dimers, trimers, and their analogs), norphilin A (norphil).
in A), fluorene (and derivatives (eg fluorenone, fluorenodiamine)), phenazine, phenanthridine, phenothiazine (eg chlorpromazine), phenoxazine, benzothiazole, xanthene and thioxanthene, anthraquinone, anthrapyrazole, benzothio. Pyranoindole,
3,4-benzopyrene, 1-pyrenyloxirane, benzanthracene, benzodipyrone, quinoline (eg chloroquine, quinine, phenylquinoline carboxamide), furocoumarin (eg psoralen and isopsoralen), ethidium, propidium, coralyne, and many Cyclic aromatic hydrocarbons and oxirane derivatives thereof; distamycin, netropsin
n), other lexitropins, Hoechst33
258 and other Hoechst dyes, DAPI (4 ', 6-diamidino-2-
Phenylindole), berenil, and triarylmethane dyes; aflatoxins; spermine, spermidine, and other polyamines; and triple helix formation, D-loop formation, and direct base pairing to single-stranded targets such as A nucleic acid or analog that binds by sequence-specific interactions. Derivatives of these compounds are also non-limiting examples of anchor groups, wherein derivatives of the compounds include compounds, compounds having any type of one or more substituents at any position. Oxidation products or reduction products thereof, but are not limited thereto.

Examples of linkers that may be used in the present invention include ester groups (where the carbonyl carbon of the ester is between the anchor and the sp ³ oxygen of the ester; this configuration is also referred to as “forward ester”). Referred to as “reverse ester group” (where the sp ³ oxygen of the ester is between the anchor and the carbonyl carbon of the ester), a thioester group (where the carbonyl carbon of the thioester is the anchor and sulfur of the thioester). Between, and also referred to as "forward thioester"), reverse thioester groups (wherein the thioester sulfur is between the anchor and the thioester carbonyl carbon, also referred to as "reverse thioester"), forward and reverse thionoester groups. , Forward and reverse dithio acid groups, sulfate groups, forward and reverse sulfonate groups, phosphate Group, and is a compound containing a functional group such as forward and reverse phosphonate groups, and the like. "Thioester" refers to a -C (= O) -S- group; "thionoester" refers to a -C (= S) -O- group; and "dithioacid" refers to -C (= S)-. Refers to the S-group. For groups that may be designated as “forward” and “reverse”, forward means that after hydrolysis of the functional group, the resulting acid functional group is covalently linked to the anchor moiety and the resulting alcohol functional group or thiol group. The orientation of the functional group such that the functional group is covalently linked to the effector moiety. The opposite direction is a functional group such that after hydrolysis of the functional group, the resulting acid functional group is covalently linked to the effector moiety and the resulting alcohol or thiol functional group is covalently linked to the anchor moiety. Direction.

Examples of effectors that may be used in the present invention include, but are not limited to, mustard groups, mustard group equivalents, epoxides, aldehydes, formaldehyde synthons, and other alkylators and crosslinkers. Mustard groups are defined as including monohaloethylamine or bishaloethylamine groups, and monohaloethylsulfide groups. A mustard group equivalent is by a mechanism similar to mustard (ie, by forming an aziridinium intermediate,
Or having a reacting group with or by forming an aziridine ring capable of reacting with a nucleophile) such as, for example, a monomesylethylamine group or a bismesylethylamine group, a monomesylethylsulfide group, a monotosylethylamine group or a vist A silethylamine group and a monotosylethyl sulfide group. Formaldehyde synthon is defined as any compound that decomposes to formaldehyde in aqueous solution and includes hydroxymethylamines such as hydroxymethylglycine. An example of formaldehyde synthon is US Pat. No. 4,337.
, 269 and International Patent Application WO 97/02028.

The alkylator compounds that can be used to prepare the white blood cells of the present invention are described by the general formulas I, II, and III below.

Formula I is the following, and all salts and stereoisomers thereof, including enantiomers and diastereomers:

[0128]

[Chemical 5]

[0129]   Where R₁, R₂, R₃, R_Four, R_Five, R₆, R₇, R₈, And R₉A few of
At least one is -V-W-X-E as defined below, and R₁, R₂, R₃
, R_Four, R_Five, R₆, R₇, R₈, And R₉The rest of is independent of the group consisting of
Selected: -H, -R_Ten, -OR_Ten, -NO₂, -NH₂, -NH-R_Ten, -N
(R_Ten)₂, -F, -Cl, -Br, -I, -C (= O) -R_Ten, -C (= O)
-OR_Ten, And -OC (= O) -R_Ten,   Where -R_TenAre independently H, -C_1-8Alkyl, -C_1-8Heteroalkyl
, -Aryl, -heteroaryl, -C_1-3Alkyl-aryl, -C_1-3Hetero
Alkyl-aryl, -C_1-3Alkyl-heteroaryl, -C_1-3Heteroarchi
Ru-heteroaryl, -aryl-C_1-3Alkyl, -aryl-C_1-3Heteroa
Rualkyl, -heteroaryl-C_1-3Alkyl, -heteroaryl-C_1-3Heteroa
Rukiru, -C_1-3Alkyl-aryl-C_1-3Alkyl, -C_1-3Heteroalkyl
-Aryl-C_1-3Alkyl, -C_1-3Alkyl-heteroaryl-C_1-3Archi
Le, -C_1-3Alkyl-aryl-C_1-3Heteroalkyl, -C_1-3Heteroarchi
Le-heteroaryl-C_1-3Alkyl, -C_1-3Heteroalkyl-aryl-C_{1- 3} Heteroalkyl, -C_1-3Alkyl-heteroaryl-C_1-3Heteroalkyl,
Or -C_1-3Heteroalkyl-heteroaryl-C_1-3Heteroalkyl;   V is independently -R₁₁, -NH-R₁₁, Or -N (CH₃) -R₁₁−
Where, -R₁₁Are independently -C_1-8Alkyl-, -C_1-8Heteroalkyl
-, -Aryl-, -heteroaryl-, -C_1-3Alkyl-aryl-, -C_{1 -3} Heteroalkyl-aryl-, -C_1-3Alkyl-heteroaryl-, -C_1-3 Heteroalkyl-heteroaryl-, -aryl-C_1-3Alkyl-,-ary
R-C_1-3Heteroalkyl-, -heteroaryl-C_1-3Alkyl-,-heteroa
Reel-C_1-3Heteroalkyl-, -C_1-3Alkyl-aryl-C_1-3Alkyl
-, -C_1-3Heteroalkyl-aryl-C_1-3Alkyl-, -C_1-3Alkyl-
Heteroaryl-C_1-3Alkyl-, -C_1-3Alkyl-aryl-C_1-3Hetero
Alkyl-, -C_1-3Heteroalkyl-heteroaryl-C_1-3Alkyl-, -C _1-3 Heteroalkyl-aryl-C_1-3Heteroalkyl-, -C_1-3Alkyl-f
Teloaryl-C_1-3Heteroalkyl-, or -C_1-3Heteroalkyl-hetero
Aryl-C_1-3Heteroalkyl-;   W is independently -C (= O) -O-, -OC (= O)-, -C (= S)-.
O-, -O-C (= S)-, -C (= S) -S-, -S-C (= S)-, -C (
= O) -S-,-SC-(= O)-,-OS-(= O)₂-O-, -S (= O)₂ -O-, -OS (= O)₂-, -OP (= O) (-OR_Ten) -O-, -P (
= O) (-OR_Ten) -O-, -OP (= O) (-OR_Ten) -Is;   X is independently -R₁₁-Is; and   E is independently -N (R₁₂)₂, -N (R₁₂) (R₁₃), -SR₁₂, And
And

[0130]

[Chemical 6]

[0131] Is selected from the group consisting of;   Where -R₁₂Is -CH₂CH₂-G, where each G is independently
Cl, -Br, -I, -OS (= O)₂-CH₃, -OS (= O)₂-CH₂C ₆ H_Five, Or -OS (= O)₂-C₆H_Four-CH₃And   And here, R₁₃Are independently -C_1-8Alkyl, -C_1-8Heteroalkyl
, -Aryl, -heteroaryl, -C_1-3Alkyl-aryl, -C_1-3Hetero
Alkyl-aryl, -C_1-3Alkyl-heteroaryl, -C_1-3Heteroarchi
Ru-heteroaryl, -aryl-C_1-3Alkyl, -aryl-C_1-3Heteroa
Rualkyl, -heteroaryl-C_1-3Alkyl, -heteroaryl-C_1-3Heteroa
Rukiru, -C_1-3Alkyl-aryl-C_1-3Alkyl, -C_1-3Heteroalkyl
-Aryl-C_1-3Alkyl, -C_1-3Alkyl-heteroaryl-C_1-3Archi
Le, -C_1-3Alkyl-aryl-C_1-3Heteroalkyl, -C_1-3Heteroarchi
Le-heteroaryl-C_1-3Alkyl, -C_1-3Heteroalkyl-aryl-C_{1- 3} Heteroalkyl, -C_1-3Alkyl-heteroaryl-C_1-3Heteroalkyl,
Or -C_1-3Heteroalkyl-heteroaryl-C_1-3Heteroalkyl.

A preferred composition according to Formula I is compound S-303 where V is -NH.
R ₁₁ —, R ₁₁ is —CH ₂ CH ₂ —, W is —C (═O) —O—,
X is -CH ₂ CH ₂ - is, E is a -N (R _{12) 2,} R12 is -CH ₂ CH ₂
G, and G is Cl. PCT application for sharing US98 / 00531
See also issue.

The general formula II, and all salts and stereoisomers thereof, including enantiomers and diastereomers, are:

[0134]

[Chemical 7]

Where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ are —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 10, and -O-C
(= O) are independently selected from the group consisting of -R _10, wherein -R ₁₀ is independently, H, -C _1-8 alkyl, -C _1-8 heteroalkyl,
-Aryl, -heteroaryl, -C _1-3 alkyl-aryl, -C _1-3 heteroalkyl-aryl, -C _1-3 alkyl-heteroaryl, -C _1-3 heteroalkyl-heteroaryl, -aryl -C _1-3 alkyl, -aryl-C _1-3 heteroalkyl, -heteroaryl-C _1-3 alkyl, -heteroaryl-C _1-3 heteroalkyl, -C _1-3 alkyl-aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-
Aryl-C _1-3 alkyl, -C _1-3 alkyl-heteroaryl-C _1-3 alkyl, -C _1-3 alkyl-aryl-C _1-3 heteroalkyl, -C _1-3 heteroalkyl-hetero Aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-aryl-C _1-3
Heteroalkyl, -C _1-3 alkyl-heteroaryl-C _1-3 heteroalkyl, or -C _1-3 heteroalkyl-heteroaryl-C _1-3 heteroalkyl; R ₂₀ is -H or-. CH ₃ where R ₂₁ is —R ₁₁ —W—X—E, where —R ₁₁ — is independently —C _1-8 alkyl-, C _1-8 hetero. Alkyl-,
-Aryl-, -heteroaryl-, -C _1-3 alkyl-aryl-, -C _1-3 heteroalkyl-aryl-, -C _1-3 alkyl-heteroaryl-, -C _1-3 heteroalkyl- Heteroaryl-, -aryl-C _1-3 alkyl-, -aryl-
Heteroalkyl C _1-3 -, - heteroaryl -C _1-3 alkyl -, - heteroalkyl heteroaryl -C _1-3 -, - C _1-3 alkyl - aryl -C _1-3 alkyl -,
To -C _1-3 heteroalkyl - aryl -C _1-3 alkyl -, - C _1-3 alkyl - heteroaryl -C _1-3 alkyl -, - C _1-3 alkyl - aryl -C _1-3 heteroalkyl -, - to C _1-3 heteroalkyl - heteroaryl -C _1-3 alkyl -, - C _1-3
Heteroalkyl - aryl -C _1-3 heteroalkyl -, - C _1-3 alkyl - heteroaryl -C _1-3 heteroalkyl -, or the -C _1-3 heteroalkyl - heteroaryl -C _1-3 heteroaryl Alkyl-; W is independently -C (= O) -O-, -OC (= 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) - a and; X is independently, -R ₁₁ - is it; and E is, -N ( _{R 12) 2, -N (R} 12) (R 13), - S-R 12 , and,

[0136]

[Chemical 8]

Independently selected from the group consisting of, wherein —R ₁₂ is —CH ₂ CH ₂ —G,
Where each G is independently, -Cl, -Br, -I, -O -S (= O) 2 -CH 3,
-O-S (= O) be ₂ -CH ₂ -C ₆ H ₅ or _{-O-S (= O) 2} -C 6 H 4 -CH 3,; and wherein R ₁₃ is independently _-C1-8 alkyl, _-C1-8 heteroalkyl,
-Aryl, -heteroaryl, -C _1-3 alkyl-aryl, -C _1-3 heteroalkyl-aryl, -C _1-3 alkyl-heteroaryl, -C _1-3 heteroalkyl-heteroaryl, -aryl -C _1-3 alkyl, -aryl-C _1-3 heteroalkyl, -heteroaryl-C _1-3 alkyl, -heteroaryl-C _1-3 heteroalkyl, -C _1-3 alkyl-aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-
Aryl-C _1-3 alkyl, -C _1-3 alkyl-heteroaryl-C _1-3 alkyl, -C _1-3 alkyl-aryl-C _1-3 heteroalkyl, -C _1-3 heteroalkyl-hetero Aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-aryl-C _1-3
Heteroalkyl, -C _1-3 alkyl-heteroaryl-C _1-3 heteroalkyl, or -C _1-3 heteroalkyl-heteroaryl-C _1-3 heteroalkyl.

The general formula III, and all salts and stereoisomers thereof, including enantiomers and diastereomers, are:

[0139]

[Chemical 9]

[0140] And   Where R₄₄, R₅₅, R₃, R_Four, R_Five, And R₈At least one of:
V-W-X-E, and R₄₄, R₅₅, R₃, R_Four, R_Five, And R₈The rest of
-H, -R_Ten, -OR_Ten, -NO₂, -NH₂, -NH-R_Ten, -N (R_Ten)₂
, -F, -Cl, -Br, -I, -C (= O) -R_Ten, -C (= O) -OR_Ten , And -OC (= O) -R_TenIndependently selected from the group consisting of   Where -R_TenAre independently H, -C_1-8Alkyl, -C_1-8Heteroalkyl,
-Aryl, -heteroaryl, -C_1-3Alkyl-aryl, -C_1-3Heteroa
Rukiru-aryl, -C_1-3Alkyl-heteroaryl, -C_1-3Heteroalkyl
-Heteroaryl, -aryl-C_1-3Alkyl, -aryl-C_1-3Heteroal
Kill, -heteroaryl-C_1-3Alkyl, -heteroaryl-C_1-3Heteroal
Kill, -C_1-3Alkyl-aryl-C_1-3Alkyl, -C_1-3Heteroalkyl-
Aryl-C_1-3Alkyl, -C_1-3Alkyl-heteroaryl-C_1-3Alkyl
, -C_1-3Alkyl-aryl-C_1-3Heteroalkyl, -C_1-3Heteroalkyl
-Heteroaryl-C_1-3Alkyl, -C_1-3Heteroalkyl-aryl-C_1-3
Heteroalkyl, -C_1-3Alkyl-heteroaryl-C_1-3Heteroalkyl
Or -C_1-3Heteroalkyl-heteroaryl-C_1-3Heteroalkyl;   V is independently -R₁₁-, -NH-R₁₁-Or-N (CH₃) -R₁₁-In
Yes, here -R₁₁-Is independently -C_1-8Alkyl-, -C_1-8Heteroarchi
Ru-, -aryl-, -heteroaryl-, -C_1-3Alkyl-aryl-,-
C_1-3Heteroalkyl-aryl-, -C_1-3Alkyl-heteroaryl-, -C _1-3 Heteroalkyl-heteroaryl-, -aryl-C_1-3Alkyl-,-ari
Rule-C_1-3Heteroalkyl-, -heteroaryl-C_1-3Alkyl-,-hetero
Aryl-C_1-3Heteroalkyl-, -C_1-3Alkyl-aryl-C_1-3Archi
R-, -C_1-3Heteroalkyl-aryl-C_1-3Alkyl-, -C_1-3Alkyl
-Heteroaryl-C_1-3Alkyl-, -C_1-3Alkyl-aryl-C_1-3Hete
Lower alkyl-, -C_1-3Heteroalkyl-heteroaryl-C_1-3Alkyl-,-
C_1-3Heteroalkyl-aryl-C_1-3Heteroalkyl-, -C_1-3Alkyl-
Heteroaryl-C_1-3Heteroalkyl-, or -C_1-3Heteroalkyl-Hete
Lower-C_1-3Heteroalkyl-;   W is independently -C (= O) -O-, -OC (= O)-, -C (= S)-.
O-, -O-C (= S)-, -C (= S) -S-, -S-C (= S)-, -C (
= O) -S-,-SC-(= O)-,-OS-(= O)₂-O-, -S (= O)₂ -O-, -OS (= O)₂-, -OP (= O) (-OR_Ten) -O-, -P (
= O) (-OR_Ten) -O-, -OP (= O) (-OR_Ten) -Is;   X is independently -R₁₁-Is; and   E is -N (R₁₂)₂, -N (R₁₂) (R₁₃), -SR₁₂, and

[0141]

[Chemical 10]

Independently selected from the group consisting of; wherein —R ₁₂ is —CH ₂ CH ₂ —G, wherein each G is independently —Cl.
, -Br, -I, -O-S (= O) 2 -CH 3, -O-S (= O) 2 -CH 2 -C 6
H _5, or be _{-O-S (= O) 2} -C 6 H 4 -CH 3; and wherein R ₁₃ is independently, -C _1-8 alkyl, -C _1-8 heteroalkyl,
-Aryl, -heteroaryl, -C _1-3 alkyl-aryl, -C _1-3 heteroalkyl-aryl, -C _1-3 alkyl-heteroaryl, -C _1-3 heteroalkyl-heteroaryl, -aryl -C _1-3 alkyl, -aryl-C _1-3 heteroalkyl, -heteroaryl-C _1-3 alkyl, -heteroaryl-C _1-3 heteroalkyl, -C _1-3 alkyl-aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-
Aryl-C _1-3 alkyl, -C _1-3 alkyl-heteroaryl-C _1-3 alkyl, -C _1-3 alkyl-aryl-C _1-3 heteroalkyl, -C _1-3 heteroalkyl-hetero Aryl-C _1-3 alkyl, -C _1-3 heteroalkyl-aryl-C _1-3
Heteroalkyl, -C _1-3 alkyl-heteroaryl-C _1-3 heteroalkyl, or -C _1-3 heteroalkyl-heteroaryl-C _1-3 heteroalkyl.

In the above general formula I, the acridine nucleus is the anchor moiety, and is —VWX—
It is understood that the group (s) comprises a scissile linker and the E group (s) is an effector group. Similarly, in the general formula III above, the psoralen nucleus is the anchor moiety and the -VWX-group (s)
Contains a scissile linker group, and the E group (s) is an effector group. General formula II is part of general formula I.

Examples 7-12 exemplify the synthesis of these compounds to obtain the leukocytes of the invention.

In one embodiment of the invention, co-owned US Pat. No. 5,399,719.
4 '-(4-amino-2-oxa) butyl-4,5', 8 previously disclosed in US Pat.
-Trimethylpsoralen ("S-59"), the disclosure of which is incorporated herein by reference in its entirety, is also useful for the photochemical treatment of leukocyte populations.

Suitable photoactivatable compounds for use in the methods of the present invention include: Furocoumarins; Actinomycins; Anthracyclins.
Anones); Anthramycins; Benzodipyrones; Fluorenes and Fluorenones; Monostral fat blue (Fluores).
Monostral Fats Blue); Norphilin
in) A: Organic dyes; Phenanthridine (Ph)
enanthridines); phenazathionium salt (Phenazathi)
onium Salts); phenazine (Phenazines); phenothiazine (Phenothiazines); phenylazide (Phenylazi)
des); Quinolines and Thiaxatenone (Thia)
xathenes)). The preferred photoactivatable compound described herein generally refers to furocoumarin.

Psoralens and Derivatives Psoralens are planar aromatic organic compounds that belong to the group of furocoumarins. Psoralens are found in nature, especially in plants including lime, cypress, celery, parsnips and figs. In humans, psoralens have been used in photochemical treatments for the management of vitiligo, psoriasis and fungal mycetoma. For reviews of psoralen photochemistry, see Parsons, B .; J. Photochem. Ph
otobiol. 32: 813-821, 1980. The basic structure of psoralen is shown below.

In particular, the invention relates to psoralen, ie [7H-furo (3,2-g)-(1)-
Benzopyran-7-one, or b-lactone of 6-hydroxy-5-benzofuranacrylic acid], which is linear:

[0149]

[Chemical 11]

And the two oxygen residues associated with the central aromatic moiety have a 1,3 orientation, and further the furan cyclic moiety is attached to the 6-position of the bicyclic coumarin system. Psoralen derivatives are derived from the substitution of linear furocumarine at the 3,4,5,8,4 ', or 5'positions.

With regard to safety issues, psoralen is already included in our daily diet. Psoralen is relatively inactive in the absence of long wavelength UVA irradiation. The effective half-life of UVA-activated psoralen is measured in milliseconds. Any drug remaining after irradiation returns to its inactive state, thus limiting side effects.

Previous protocols for the inactivation of psoralens have been devoted to the removal of molecular oxygen from the reaction before and during exposure to light to prevent oxygen radicals generated during irradiation from damaging blood products. Needs removal. L. Lin et al., Blood 74: 5.
17 (1989); U.S. Pat. No. 4,727,027, Wiesehahn. The method of the invention can be used to inhibit the proliferation of white blood cells in the presence of oxygen. Furthermore, it is not necessary to reduce the concentration of molecular oxygen with the novel psoralen used in the method of the present invention. Preferred psoralens have low mutagenicity and high nucleic acid binding affinity.

8-Methoxypsoralen (known in the literature by various names, eg xanthotoxin, methoxsalen, 8-MOP) is a naturally occurring psoralen with low mutagenicity in the Ames assay. .

4'-Aminomethyl-4,5'8-trimethylpsoralen (AMT) is one of the most reactive nucleic acid-bound psoralen derivatives, providing up to 1 AMT adduct per 3.5 DNA base pairs. Do (ST Isaacs, G. Wieseha
hn and L.L. M. Hallick, NCI Monograph 66:21
, 1984).

“4′-Primary amino-substituted psoralen” or “4-PAP” is a psoralen compound having an NH ₂ group attached to the 4′-position of the psoralen by a hydrocarbon chain having a total length of 2-20 carbons. Where 0 to 6 of these carbons may be independently substituted by NH or O, and each position of substitution is separated from each other by at least 2 carbons. , And is separated from the psoralen by at least one carbon. The 4′-primary amino-substituted psoralen is a further substituent at the 4,5′- and 8-positions of psoralen.
n), which substituents include, but are not limited to, the following groups: H and (CH ₂ ) _n CH ₃ (where n = 0-6).

“5′-Primary amino-substituted psoralen” is also referred to herein by the abbreviation “5-PAP”.
5 of psoralen by a hydrocarbon chain having a total length of 1 to 20 carbons.
Is defined as a psoralen compound having an NH ₂ group attached to the'position, wherein 0-6 of these carbons are independently replaced by NH or O, and each position of substitution is at least 2 Of carbons are separated from each other in the position of substitution and at least one carbon is separated from the psoralen. 5'-primary amino substituted psoralens may have additional substituents at the 4,4 ', and 8 positions of the psoralen, the above substituents, the following substituents: H and (CH _{2) n} CH ₃ ( Here, n = 0 to 6) can be mentioned, but the invention is not limited thereto. 4-PAP and 5-
Examples of PAPs (including S-59) and methods for their synthesis are described in US Pat. No. 5,585.
No. 5,503; No. 5,578,736; No. 5,556,993 and No. 5,399,719.

Preferred psoralens include 5′-1 primary amino-substituted psoralen and 4′-1 primary amino-substituted psoralen (collectively referred to herein as PAP), 8-methoxypsoralen (8-MOP), 4'-aminomethyl 4,5 ', 8-trimethylpsoralen (AMT), 5-methoxypsoralen (5-MOP), and trioxalen 4,5'8-trimethylpsoralen. AMT, 5-MOP, 8-
MOP and trioxalen are commercially available.

The most preferred psoralen is S-59 having the formula shown above (see abbreviation S-59 below). S-59 is a synthetic psoralen that can reversibly bind nucleic acids by intercalation. Upon irradiation with UVA, intercalated S-59 forms monoadducts and interstrand crosslinks with RNA and DNA. S-59 photochemical treatment is specific for nucleic acids, and S-59 readily penetrates cell and nuclear membranes. S-59 combines high water solubility and nucleic acid binding affinity with high quantum yield for the formation of nucleic acid adducts. This allows the use of lower psoralen concentrations in PCT to inhibit proliferation in the absence of non-specific cell damage. Mutagenicity and genotoxicity studies on S-59 demonstrate a 40,000-67,000-fold safety margin of the concentration used to scrub pathogenic agents in blood products. Finally, P
The dose of S-59 used for CT is the daily dietary intake of chemical groups containing furocumarine, a psoralen (Wagstaff, DJ Reg. Tox).
． Pharm. 14: 261-272, 1991) is sufficient.

In one embodiment, the cell population, including the leukocyte population, is treated with a photoactivatable compound that, upon photoactivation, comprises a moiety capable of forming a covalent bond with a nucleic acid.
Therefore, in order to form a covalent bond with a nucleic acid, the photoactivatable compound must be activated by light. The light is ultraviolet light (UVA, UVB, UVC
) And visible light. In one embodiment, the wavelength used for PCT is in the range 200-450 nm. In a preferred embodiment, the light used for PCT has a wavelength of 320-400 nm.

Preferably, the cell population is photochemically treated with UV light having a wavelength in the range 320-400 nm. UVA irradiation alone induces minimal damage to cells.

In one embodiment, the photoactivatable compound comprises a psoralen molecule. PCTs with appropriate psoralen concentrations and UVA light doses, for example, promote the destruction of diseased cells, infected cells, or virulence factors, induce anti-leukocyte effects, and facilitate transplantation by a second population of cells. Growth inhibition is obtained while effectively maintaining leukocyte immunogenic function for treatment of mixed chimerism, promoting immune reconstitution, facilitating immune reconstitution, and treating mixed chimerism. Psoralen reactivity is not only specifically directed to nucleic acid-to-protein in the cell, but also due to differences in nucleic acid structure that allows better intercalation into DNA. Binding affinity (Cimino, GD et al., Ann. Rev. Biochem. 5).
4: 1151-1193, 1985). In addition, this technique can be used to differentially modify transcriptionally active (as opposed to inactive) genes due to different conformations and states of histone binding that affect psoralen binding. May be possible. Treatments such as PCT therefore have the ability to interfere with cellular function (mainly at the level of DNA synthesis (replication)) with less effect on transcription, and minimal effect on protein synthesis. Or has no effect.
In the case of PCT, for example, the level of interference depends on the concentration of psoralen and the UV used.
A light dose.

A sample of leukocytes mixed with a photoactivatable compound is photochemically treated using a photoactivatable device. In general, suitable photoactivatable devices for this method of PCT include: (a) means for providing electromagnetic radiation of a suitable wavelength to induce photoactivity of the photoactivatable compound; b) means for supporting a plurality of samples in fixed association with irradiation providing means during photoactivation; and (C
) A means for maintaining the temperature of the sample within the desired temperature range during photoactivity.

In one embodiment, the photoactivating device is between 200 nm and 450 nm,
It is possible to emit a spectrum of a given intensity of electromagnetic radiation, which preferably comprises wavelengths between 320 nm and 400 nm. Specific wavelengths can be selected using appropriate filters in the photoactivation device. Suitable photoactivatable devices and methods of photoactivating photoactivatable compounds by use of the device are described in US Pat. No. 5,184,184.
, 020 and 5,503,721, both of which are described in Hearst.
And disclosed in WO 96/39820. Other photo-activated devices that can be used are General Electric F20T12-BLB type fluorescent UVA bulbs (Alter, HJ, et al., The Lancet, 24: 1.
446 (1988)), and P. W. Allen Co. , Type A405-TLGW / 05 long wavelength UV lamp manufactured by London, Inc.

Photochemical Treatment of Leukocytes with Psoralen Stock solutions of psoralen in water or a suitable solvent are prepared as illustrated in the Examples below.

A stock solution of psoralen is added to a suspension of purified donor leukocyte populations to the desired final concentration. Units treated with 8-MOP are P saturated with 8-MOP.
Prepared in the above-described preparation procedure using AS. The purified donor leukocytes are
Irradiate with UVA light to a final dose of 10 ^{−3 to} 100 J / cm ^{2 in a} photoactivation device. The sample is shaken during irradiation. A white blood cell sample is removed as a control before irradiation.

Typically, PAP is added to the purified leukocyte preparation at a concentration of between 10 ^{−4 and} 150 μM, preferably between 10 ^{−3 and} 150 μM. White blood cells are generally between about 10 and 10 ⁹ cells / ml, preferably between about 10 ^{3 and} 10 ⁸ cells / ml.
Even more preferably between about 10 ^{4 and} 10 ⁷ cells / ml, most preferably about 2 ×.
It is at a cell density of 10 ⁶ cells / ml.

In a preferred embodiment, PCT is administered to leukocytes in combination with S-59 at 320
It is performed using UV light with a wavelength between ˜400 nm. Light activation process 3
Limiting to wavelengths greater than 20 nm minimizes direct nucleic acid damage. This is because absorption above 313 nm is negligible by nucleic acids. The cells are preferably about 10 ^{-3 to} 100 J / cm ² , more preferably 1 to 10.
It receives a quantity of light in the range of ³ J / cm ² . In the most preferred embodiment, the light quantity is 3 J / cm ² . The irradiation is generally at a light density of between 1 and 50 mW / cm ² .
The time of exposing the white blood cell sample to UV light is between about 1 second and 60 minutes, preferably about 3 minutes, more preferably about 1 minute.

After PCT, the leukocyte sample was prepared as described above and in the Examples.
Proliferated and assayed for potency involved in leukocyte activity. Temporarily,
The following parameters are measured: cell viability; proliferative activity; cytokine secretion; and surface antigen expression.

Therapeutic Application The treated leukocyte populations of the invention are non-proliferative but retain immunological activity,
It has a variety of prophylactic and therapeutic uses, as described below. Typically, individuals undergoing DLI comprising the treated leukocyte population include, but are not limited to, transplant patients such as patients following BM transplant and patients who are scheduled for solid organ transplant. The fact that treated leukocytes do not elicit GVHD allows for higher numbers of leukocytes (ie, higher cell doses) to be administered in the DLI,
The effect of treatment is maximized.

Treated leukocytes are donor leukocytes, particularly for immunizing immuno-compromised patients such as allo (allogeneic) BMT patients or in elderly patients suffering from CLL. Useful for injection (DLI). DLI with treated leukocytes is useful for alleviating a variety of conditions, including: Chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML). ), Acute myelogenous leukemia (AML), and leukemias including acute lymphoblastic leukemia (ALL); multiple myeloma (MM); non-Hodgkin's and Hodgkin's lymphoma; large cell lymphoma (LCL); and Epstein- Bar virus-induced lymphoproliferative disorder (EBV-BLPD or EBV-induced lymphoma)
Solid tumors, including breast cancer, lung cancer, ovarian cancer, testicular cancer, prostate and colon cancer, melanoma, renal cell tumor, neuroblastoma, head and neck tumors; aplastic anemia; myelodysplasia; immunity Deficiencies, eg, unclassifiable, SCID, Viscott-Aldrich syndrome, granulocytopenia; mixed chimerism; and genetic disorders.

[0171]   DLI is useful in a variety of formats for prophylactic and therapeutic purposes: (A) Adoptive immunotherapy for allogeneic transplantation; (B) immune reconstitution for immunosuppressed or immunodeficient patients; (C) Treatment of elderly leukemia patients ineligible for transplantation; (D) BMT light or mini transplant.

The BMT light protocol is a two-step process that includes: i) minimal conditioning regimen and induction of transplant tolerance with donor stem cells; and ii) DLI.
Immunotherapy using repeatedly. This protocol avoids the toxicity of chemotherapy regimens. The BMT light protocol is appropriate in the following indications: patients who are usually not eligible for transplantation, especially in elderly (> 65 years); and most of them, CLL patients, who are severely immunocompromised To BMT light cure reduces toxicity, GVHD and infection. An example of a BMT light protocol is described in Example 13 below. (E) CD34 selection / T cell depleted allograft with concurrent DLI.

DLI provided at the same time as CD34 + selected cells (selection to enrich for stem and progenitor cells) is CMV, Epstein-Barr virus (EBV), adenovirus (Ad), Kaposi's sarcoma associated with herpes virus. , And to confer adoptive immunotherapy against infections such as fungal infections, and leukemias, eg CM
Useful for treating L. (F) Treatment of mixed chimerism. (G) Rescue from chemotherapy or chemoresistant leukemia.

In one embodiment of the invention the PA-DLI is haploid identical.
Identical) (haplo) transplant. Haplo transplantation usually requires near complete T cell depletion, resulting in severe and persistent immunosuppression. PA
-DLI is useful for maintaining immunocompetence in the host and may confer GVL effects for the treatment of cancer.

In a preferred embodiment, the leukocytes of the invention that are not functional in GVHD but retain some immune function are used in DLI to alleviate recurrent cancers or (especially CML and acute leukemia, lymphoma). And in multiple myeloma) to eliminate the slight residual disease after allogeneic BMT.
A large number of donor CTLs, specific for the bcr-abl protein of CML, contribute to the efficacy of DLI in recurrent CML. A cancer patient is determined to be "recurrent" if malignant cells are detected by the physician by the means customary for monitoring the presence of cancer cells.

In another embodiment, DLI comprising non-proliferating leukocytes is used to avoid destructive regimens on the bone marrow in the treatment of leukemia patients during induction chemotherapy (first round of chemotherapy). It is used to eliminate residual disease. Using DLI in this setting can reduce the toxic dose of chemotherapeutic agents administered during induction.

In one embodiment of the invention, PA-DLI may also be administered as a solid organ transplant (eg, kidney, heart, Skin). Most of the organ rejection in allogeneic transplantation results from the presence of passenger leukocytes in the donor organ. Due to its lack of proliferative capacity and the inability to mediate GVHD, DLI (using leukocytes from organ donors) was used prior to transplantation because of suppression of the immune response that would otherwise induce organ rejection. It can be used as a tolerizing regimen. Tolerization of the host for solid organ transplantation consists of: a certain level of bone marrow destructive regimen to allow survival of donor cells in the circulatory system before or during the organ transplant procedure. Infusion of PA-DLI with or without donor stem cells, in combination. D
LI may facilitate seeding of donor stem cells in this setting, taking into account mixed chimerism in the host.

In all types of transplant and transplant situations, the treated leukocytes can be administered prior to, concurrently with, or after the transplant. At any of these time points, multiple injections of the treated leukocyte population can be made.

Non-proliferative, immunofunctional leukocytes are particularly valuable for DLI in patients who are contraindicated in traditional allogeneic BMT because of old age or comorbidities. Older patients (> 65 years) who present with cancer are not the top priority candidates for allogeneic BMT. The reason for this is in part because transplantation cannot reconstitute the old immune system very well, and the procedure is harsh. Currently, the only option available for cancer control in elderly patients is chemotherapy, whose side effects may be unacceptable to these patients. The treated leukocytes of the present invention provide a much sought-after alternative for the treatment of cancer in elderly patients and for conferring immune protection against opportunistic infections. DLI is useful for destroying cancer cells without being associated with GVHD.

In certain cases of treatment, it may be desirable to expand a subpopulation of donor leukocytes, particularly antigen-specific T cells and progenitor cells, prior to subjecting the donor leukocyte population to treatment with the compound. The leukocyte population can be stimulated with an antigen specific to the diseased cell (eg, a tumor-associated antigen) or a virulence factor to increase / enrich for the number of cytotoxic helper T cells specific to that antigen. This stimulation can be done in vivo by vaccinating the donor with the appropriate antigen prior to isolation of leukocytes from the stimulated donor. Alternatively, it may be performed in vitro prior to treatment of the leukocyte population with the compound.

For example, IgG vaccinated donors from patients with multiple myeloma have high levels of the myeloma-specific precursor CTL. Therefore, it is preferred to prepare leukocytes from so vaccinated donors for use in the treatment of multiple myeloma (MM). For immunization of patients with non-Hodgkin's lymphoma, pulsing dendritic cells with tumor-associated antigens has been described by Hsu, FJ et al., Nat. Med. 2: 52-58
, 1996. The technique of pulsing dendritic cells with peptides / antigens is
Stingl, G et al., Immunol. Today, 16: 330-333, 1
995. CDNA immunization (transfecting dendritic cells with the appropriate cDNA) is described by Pardoll, D et al., Immunity 3:16.
5-169, 1995. In adoptive immunotherapy for the treatment of CML, the donor may be vaccinated with the bcr-abl peptide prior to isolation and subsequent infusion of donor leukocytes for treatment by the methods of the invention. Effective immunogenic peptides for vaccination are described, for example, by Ten Bosch, G et al., B
Loud, 88: 3522-3527, 1996. In yet another example, donor leukocytes for the treatment of prostate cancer are primed with prostate specific antigen (PSA), either in vivo or in vitro, prior to treatment with the methods of the invention. Such vaccination may be applied to increase the amount of precursor T cell populations and / or T cell populations specific for other tumor-specific antigens, viral antigens and antigens of other pathogenic agents. .

Leukocytes from donors vaccinated with the appropriate antigens are used in opportunistic infections in immunocompromised patients (eg cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpesvirus-associated capsocial virus). It is useful prophylactically or therapeutically to confer immune protection against sarcomas, or adenovirus (Ad) infections. For example, a donor for vaccination with CMV antigen and white blood cells isolated and D for treatment of patients exhibiting CMV infection as a result of immunosuppression.
For use in LI, it may be subjected to treatment according to the methods of the invention. Immunocompromised patients include those undergoing treatment with immunosuppressive drugs such as organ transplant patients (BM and other organs), HIV or EBV infected patients, cancer patients, and patients with immunodeficiency diseases. included.

Donor leukocytes can also be stimulated in vitro to increase precursor pools (also called ex vivo expansion). For example, in the case of multiple myeloma, donor leukocytes can be stimulated by in vitro culture with patient-specific MM antigens presented by dendritic cells prior to treatment to block GVHD activity.

Ex vivo or in vitro stimulation also has anti-leukemic T for the treatment of CML.
It can be used to expand the population of cells. In CML, the c-abl proto-oncogene on chromosome 9 has a breakpoint cluster region (bcr) on chromosome 22.
A classical translocation to (t (9; 22) (q34; q11)) results in the bcr-abl fusion gene, expressing the bcr-abl210 kD fusogenic oncoprotein. The bcr-abl fusion protein is CML-specific, and bcr-abl
The two major variants of the protein are well characterized antigens. bcr-a
In vitro stimulation to expand T cells specific for the bl protein is described, for example, by Choudhury, A et al., Blood, 89: 1133-1142, 199.
7; ten Bosch, G et al., Blood, 88: 3522-3527, 19
96; and Mannering et al., Blood, 90: 290-297, 19;
It can be carried out as described in 97. Peptides representing breakpoint regions or junctions (novel junction amino acid sequences) can serve as immunogens for in vitro immunization of human T cells from healthy donors (ten Bosch et al., 1996, supra; Ma.
nnering et al., 1997, supra). The resulting CD4 ⁺ T cells recognized bcr-abl expressing cells from allogeneic CML patients. In another approach, dendritic cells (DCs) can be generated in vitro from peripheral blood cells of CML patients and used as antigen presenting cells for ex vivo expansion of anti-leukemic T cells (Choudhury et al., 1997, supra). More generally, donor antigen-presenting cells (APCs) can be pulsed with antigens characteristic of diseased cells or co-cultured with diseased cells (eg, inactivated tumor cells). These APCs are then exposed to donor leukocytes, which are then infused into the recipient. Or donor leukocytes and diseased cells (or diseased cell antigens)
Direct contact with can be used to expand the T cell population in donor lymphocytes.

The “antigen” used to expand T cells can be a polypeptide, lipid, or carbohydrate moiety (or any combination, such as a glycoprotein), and isolated from diseased cells or virulence factors. Or recombinantly produced. The polypeptide-containing antigen need not comprise the full-length protein as long as it contains at least one epitope. The antigen may be included in the vaccine composition as a full length protein or a fragment thereof, or as a recombinantly produced fusion protein, and may or may not be conjugated to an adjuvant, or otherwise adjuvanted with an adjuvant. It may or may not be presented. The vaccine can take a variety of forms: diseased cells expressing the antigen, or cell membrane preparations thereof; or virulence factors (preferably in inactivated form). Methods for preparing vaccines are described in the literature. See, eg, Harlow, supra. Methods of immunization and in vitro (ex vivo) stimulation and expansion of precursor cell populations are known in the art.
For example, Choudhury, A et al., Blood, 89: 1133-1142,
1997; ten Bosch, G et al., Blood, 88: 3522-3527.
, 1996; and Mannering et al., Blood, 90: 290-297.
, 1997.

For in vivo use, leukocytes are generally administered in plasma, synthetic media, or other physiological buffer solutions at a dose of about 10 ⁵ -10 ¹¹ leukocytes per kg body weight per infusion, about 50-500 ml. Or it is administered in a larger volume. These parameters may vary with treatment and disease and are determined by the physician treating the patient. According to standard DLI practice, leukocytes are present at 10 ⁷ -1 per kg body weight.
It is administered in an amount of 0 ^8. DLI can be given with chemotherapy.

[0187]   For in vivo use, the leukocytes of the invention are generally administered intravenously.

GVHD usually appears within 90 days after BMT. To alleviate the disease in patients with recurrent cancer, DLI is preferably performed within 1 week to 3 months after the recurrence is diagnosed. DLI for adoptive immunotherapy after allogeneic BMT
Can be delivered as maintenance therapy from before BMT to years later. For cancer patients who receive DLI instead of BMT, DLI is preferably administered promptly after the cancer has been diagnosed. In the absence of BMT, DLI may be provided prior to, during, or after chemical or irradiation or other therapeutic regimen. The appropriate timing of DLI for all indications is determined by a physician skilled in treating the disease.

Assessment of Treatment Success: Following DLI, the host or patient will follow GVHD according to conventional procedures.
Being monitored for. GVHD usually appears within 90 days after infusion. Clinical GVHD symptoms include skin rash, swelling and damage to the liver, digestive tract, lungs and joints, severe diarrhea and jaundice. Patients' levels of bilirubin and liver function enzymes can be measured, and liver biopsies can also be performed. Patients undergoing DLI
Detailed clinical history, physical examinations, general study evaluations can be monitored regularly, including complete blood counts and differentials, urinalysis, blood urea nitrogen creatinine, bilirubin, aspartate transaminase (AST), alanine transferase (ALT). , Alkaline phosphatase, Na ⁺ , K ⁺ , Cl ⁻ , albumin, total protein, glucose, and radiography. The cancer situation is, for example,
It can be assessed by bone marrow aspirate and biopsy examination, cytogenetic examination (including immunohistochemical analysis), and molecular analysis.

For the purposes of the present invention, a recipient of treated leukocytes, if there is a visible or measurable improvement in the symptoms of the disease or the symptoms resulting from the disease,
It is assumed that the disease (the disease means a malignant disease or infection) has been alleviated. Methods for assessing symptoms and their improvement vary with the disease state, but are well known to clinical technologists. One useful endpoint for assessment of treatment success is 10.
The mortality rate on day 0.

The methods and compositions of the invention are also useful for the prevention of GVHD in situations other than DLI. These include, but are not limited to, the infusion of platelets and the febrile, non-hemolytic infusion reaction that occurs as a result of cytokine accumulation during platelet storage.

The methods of the invention, and the treated non-proliferating leukocytes of the invention, also have in vitro uses. For example, the PCT procedure is a minimally invasive method that inhibits DNA synthesis in competent primary cultures or cell lines (eg, progenitor cells and stem cells) without affecting cell biosynthesis. Therefore, in an assay to test whether cell differentiation or developmental stage is dependent on proliferation / DNA synthesis,
Treatment conditions can be used to prepare isolated stem cells, leukocytes or other cell lines. Such studies may identify target molecules along a differentiation or developmental pathway,
And it may facilitate the development of agents to inhibit or promote differentiation. Treated leukocytes can also be used, for example, as stimulator cells in a one-way MLR assay to stimulate the immune response of proliferative allogeneic cells without the stimulator cells themselves proliferating simultaneously. PCT and related treatments replace the current irradiation or use of mitomycin C to achieve cytostatics of stimulated cells with compounds capable of forming covalent bonds with DNA. For these in vitro uses, the composition of treated leukocytes or other cells may be provided alone or as part of an assay kit. The assay kit can be for MLR or for a differentiation assay. For these purposes, treated cells are generally provided in frozen form. Alternatively, the kit may provide reagents and instructions for the preparation of white blood cells or other cells having the above characteristics. Reagents include one or more compounds capable of forming a covalent bond with DNA. For example, the kit used for PCT comprises one or more photoactivatable compounds, preferably PAP or acridine, more preferably S-59.

The following examples are intended to illustrate the invention described herein and not to limit its scope. Certain modifications of the method will be readily apparent to those skilled in the art and are covered by the claims.

Examples Various Assays (Proliferation Assay with 3H Thymidine Incorporation) In a representative assay, cells are placed in a 96-well plate for the desired time and the test compound.
Grow in the presence or absence (eg, DNA replication inhibitor or mitogen). The medium is typically prepared to contain 1 Ci / 50 μl / well with about 20 Ci / mmol specific activity of 3H thymidine. The medium / label is added to the cells and the cells are incubated at 37 ° C for about 4-6 hours or longer. Briefly, cells are transferred to a paper filter disk using a multichannel cell harvester and the free (unincorporated) label is washed away. The disc is then dried, transferred to a vial and immersed in scintillation fluid. The vials can then be placed in an automated counter, counted for the entire tritium range, and the average counts per minute (cpm) determined for 3 samples.

(Measurement of Compound-DNA Adduct) The degree of DNA modification was measured using 3H-labeled PAP. PAP was added to the desired final concentration in the purified leukocyte sample. A 20 mL aliquot in a mini PL 2410 plastic container was illuminated with the appropriate amount of UV light. Control samples were treated with PAP alone without UVA. After irradiation, leukocyte DNA was purified. The DNA content of each sample was determined by measuring the absorbance at 260 nm. The number of psoralen adducts per 1000 base pairs (bp) was calculated from the radioactivity in the DNA sample. The standard curve correlates the amount of 3H counts with the number of adducts.

PCR Inhibition Assay The polymerase chain reaction (PCR) methodology is well known in the art. K. K., incorporated herein by reference. B. Mullis et al., US Pat. No. 4,683,1
95 and 4,683,202. Using this assay, P
The effect of AP-DNA adducts on the template function of particular nucleic acid sequences can be evaluated.

DNA samples from platelet concentrates photochemically treated or gamma-irradiated (
1 μg) for the 242 bp sequence in the HLA-DQα locus, or β
PCR amplified for the 439 bp sequence in the globin locus. Control(
Untreated DNA was serially diluted (1:10) and then amplified. The treated DNA was amplified without dilution. PCR amplification was performed for up to 35 cycles. This assay
It provided a means to associate functional T cell proliferation inhibition with direct modification of nucleic acids as measured in cell proliferation assays.

Examples 1 to 4 below show that the effect of PCT on treated leukocyte properties was
It is shown to be associated with NA modification and inhibition of polymerase activity. Second, PCT treatment of leukocytes in platelet concentrates was associated with transfusion-activated GVH in a mouse transfusion model.
Can interfere with D.

Example 1 Photochemical inactivation of T cells Example 1 showed that leukocytes can be inactivated by PCT in a dose-dependent manner. S-59, AMT and 8-MOP were used as exemplary psoralens. PC
The dose dependence of T varied with the properties of the psoralen used, with S-59 tested as the most effective.

S-59 (square), 4′-aminomethyl 4,5 ′, 8-trimethylpsoralen [AM
The results characterizing the dose-related effects on white blood cells in platelet concentrate (PC) of photochemical treatment with T] (triangles) and 8-methoxypsoralen [8-MOP] (circles) are shown in Figure 1. . Platelet concentrates are described in US Pat.
Prepared and processed as described in 593,823. White blood cells from photochemically treated and untreated pooled random donor PCs were plated in the LDA assay. Three psoralens of various concentrations, 1 Joule / cm2
Used with a constant light intensity of UVA. T cells were 0.05 μM S-59, 1.
It was inactivated with 0 μM AMT, and 10.0 μM 8-MOP to the limit of detection by LDA (indicated by arrow). These data clearly show that S-59 is a superior photoreagent compared to AMT and 8-MOP in inhibiting T cell proliferation.

Example 2 Mixed Leukocyte Reaction Peripheral blood was drawn from 4 individuals into anticoagulant citrate dextrose (ACD) tubes. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Ficoll. PBMCs from 3 donors were pooled and allogeneic stimulators (
Allostimulator). PBMCs from the rest of the individuals were used as responders. Stimulators and responders PBMC were treated with RPMI (10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, and 100 μl).
g / mL streptomycin supplemented) and resuspended in this supplemented medium.

Stimulator PBMCs were treated with 2500 cGyγ from Blood Bank 137 Cesium Irradiator.
Exposed to actinic radiation. Stimulator cells were then placed in a 96-well round bottom plate at 1.0.
Plated in 100 μL aliquots containing × 10 5 cells.

The responder cells were subjected to PCT with S-59 as follows. Responder cells were resuspended in 30 mL of the above cell culture medium, and S was added to the cell suspension.
An aqueous -59 stock solution (dissolved in water) was added to the final concentration shown in Figure 2, Figure 3, or Figure 4. The concentration and volume of S-59 stock solution used was the final S desired.
It was changed depending on the −59 concentration. Then 30 mL of cell solution containing S-59 was transferred to a small PL2410 bag and irradiated with UVA light at a dose of 3 J / cm 2. For FIG. 3, the light intensity was 3.0 J / cm 2. The cells are then centrifuged, resuspended in cell culture medium, and 100 μl containing 1.0 × 10 5 cells.
Plated in L aliquots and mixed with stimulator cells in a final volume of 200 μL per well.

The plates were then incubated at 37 ° C under 5% CO2. Supernatant samples from MLR were removed after 1, 2 and 7 days of incubation and stored at −80 ° C. for subsequent cytokine analysis. Wells designed for measuring cell proliferation were incubated for 6 days. After this 6 day incubation,
All wells were used to measure proliferation of cells receiving 1 μCi of 3H thymidine. After 24 hours of incubation in the presence of 3H thymidine, cells were harvested on glass fiber paper. The amount of 3H thymidine incorporated was then quantified by liquid scintillation counting. The results of 3H thymidine incorporation by responder cells photochemically treated with various concentrations of S-59 are shown in FIG.

Analysis of the synthesis of cytokines IL-2 and IFN-γ by treated cells was analyzed by R & D.
The assay was performed using the ELISA assay kit provided by D systems according to the manufacturer's instructions. 3 and 4 show the results of IL-2 and interferon-γ production, respectively. In FIG. 4, IFN-γ analysis was performed on cell culture medium collected on day 7.

Example 3 Modulation of Leukocyte Function by PCT Example 3 demonstrates that leukocyte activity, when measured by cytokine synthesis and surface marker expression (exemplified by CD69 lymphocyte activation marker expression)
It was shown that it can be adjusted by the concentration and light quantity of the psoralen used for CT. This data titrates UVA light dose and concentration of photoactivatable compounds to block proliferation of T cells in leukocyte populations and to maintain leukocyte activity, as indicated by cytokine synthesis and surface marker expression. Provide evidence that a suitable range can be obtained that is effective for both.

A. Modulation of cytokine synthesis by PCT IL-8 synthesis was also measured using different concentrations of S-59 at a constant light intensity of 0.5 J / cm2 UVA (see Figure 5). Concentrations of S that increase IL-8 synthesis
It has been found that it can be adjusted by -59 and PCT. Similar behavior is S-59
Obtained at 1.0 J / cm2 over a range of concentrates (data not shown). These results indicate that cytokine synthesis can be modulated by the concentration of psoralen used for PCT and the dose of light.

B. Modulation of CD69 expression on leukocytes after PCT treatment Induction of CD69 expression by leukocytes upon their activation by PMA and calcium ionophore, the use of fluorescent antibodies against CD69 and FACS-.
Determined by Scan analysis (see Figure 6). Expression of CD69 was measured as a function of time after photochemical treatment with different concentrations of AMT and 1 J / cm2 UVA. The effect of PCT on induction of CD69 expression was also found to be psoralen dose-dependent, with expression of CD69 showing greater inhibition at increasing concentrations of AMT. At the low doses expected for S-59 in this study, induction remains intact.

Example 4 DNA Modification by PCT Example 4 shows that the effect of PCT on the properties of treated leukocytes was
It was shown to correlate with modification and inhibition of polymerase activity.

Psoralen adducts on leukocyte genomic DNA were pooled in random donor PC
After photochemical treatments in S., 3H radiolabeled S-59, AMT, and 8-MOP plus 1.9 J / cm2 UVA were used and measured by scintillation counting the isolated DNA of treated leukocytes (FIG. 7). 150 μM S
Photochemical treatments with -59, AMT, and 8-MOP were performed respectively in 12.
0, 6.0, and 0.7 adducts / 1000 bp DNA were induced. The relative number of adducts on the genomic DNA of treated leukocytes after PCT correlated with the effect that PCT had on leukocyte function, and showed that this effect was a direct result of DNA modification. Inhibition of DNA amplification by DNA polymerase in the PCR inhibition assay (data not shown) showed the same dependence on the concentration of psoralen used during PCT.

Results from all of the biological and molecular parameters used herein to study the effect of PCT on white blood cells in vitro are consistent. Assessment of leukocyte function by limiting dilution analysis, measurement of cytokine production, and psoralen-induced DNA
Quantification of damage showed that PCT inactivates leukocytes in a dose-dependent manner. With the proper dose, leukocyte function such as cytokine production or expression of antigenic markers can be controlled. Furthermore, of the three psoralens tested, S-59
Are most effective at inactivating white blood cells. The dose range in which S-59 inhibits T cell proliferation is approximately 3.5 log units. Thus, the dose window in which leukocyte proliferation and associated GVHD can be inhibited but immune function retained is greater than that provided by other means of inactivation.

Example 5 Prevention of TA-GVHD in Mouse Infusion Model The following mouse infusion model is a well-characterized model that stimulates the human clinical syndrome of TA-GVHD (Fast, LD et al Blood 82: 292,1
993). Based on the promising in vitro results, the effect of S-59 photochemical treatment on the inhibition of TA-GVHD was examined using this in vivo model with homozygous parent.
Splenocytes from (A strain, H-2a) were evaluated by infusion into immunocompetent heterozygous F1 hybrid recipient mice (B6AF1 strain, H-2a / b).

About 108 donors (A or F1) via lateral tail vein to each recipient (F1)
Splenocytes were infused. Splenocytes obtained from density gradient centrifugation were used as a source of viable T cells. Three infusion groups were used: (1) Handling control group (F1 → F1)
: B6AF1 splenocytes were infused into B6AF1 recipients; (2) Positive T
A-GVHD control group (A → F1); donor A splenocytes were infused into B6AF1 recipients; (3) S-59 photochemical treatment group (PCT A → F1); donor A
Splenocytes were treated with 150 μM S-59 and 3 Joules / cm 2 UVA and then infused into B6AF1 recipients.

Recipients were monitored for biological evidence of TA-GVHD for 2 weeks post transfusion. Recipient spleens were analyzed for donor T cell engraftment using two-color fluorescence activated flow cytometry. In this assay, spleen T cells were pan-T cells, anti-CD3 antibody, and also anti-H-2b.
Stained with antibody. Donor A T cells were detected by their lack of reactivity with anti-H-2b antibody. In control experiments,> 99% of CD3-positive splenocytes from F1-> F1 recipients were labeled with anti-H-2b antibody. In parallel, the presence of recipient cytotoxic lymphocytes (CTL) was measured by using the 51Cr lysis assay. Splenocytes were treated with an effector: target ratio of 150: 1 at 37 ° C for 4-
Incubated with 51 Cr-labeled EL-4 cells (H-2b) for 6 hours. CTL
Cytotoxic activity was measured by target cell lysis and release of 51Cr into the culture medium.

The results show that the recipients in the control F1 → F1 group remained healthy throughout the duration of the study, while the recipients in the A → F1 group 2-3 weeks after transfusion of donor splenocytes. It has been shown to have developed the biological manifestations of TA-GVHD. TA
-GVHD was characterized in splenomegaly, recipient spleen by donor T cell (H-2a) inoculation (28.6 ± 11.3%) and presence of CTL (35 ± 18.7% 51Cr lysis) (Table 1).

Giant splenomegaly was evaluated 2 weeks after infusion by measuring the spleen: body weight ratio. The development of immunodeficiency was detected 3 weeks after infusion by assessment of thymocyte integrity. Thymoplasty is a reliable indicator of acquired immunodeficiency associated with TA-GVHD.

Recipients were also monitored for clinical symptoms of GVHD, including weight, posture, activity, skin integrity, skin structure, white blood cell count, red blood cell count, and platelet count. Every week, the body weight of healthy animals in the F1 → F1 group increased over time, but the average body weight of animals in the A → F1 group did not increase over time.

The mean clinical score of animals in the A → F1 group (grade 0-2, healthy animals were grade 0) increased over time, TA-GVHD emerged out, and progressively more severe. It was shown that. Animals in the PCT A → F1 group were control F1
→ Similar to animals in F1 group and remains healthy and visible T
There were no clinical signs of A-GVHD. When chasing for more than 10 weeks after infusion, A →
Mortality of 25% was observed in the F1 group, but control F1 → F1 and PC
All animals in the TA → F1 group remained healthy.

[0219]

[Table 1]

1. S. D. = Standard deviation 2. Measured 2 weeks after infusion 3. Measured tissue sections were prepared 3 weeks after infusion and examined for histological abnormalities after blind coding. The liver was evaluated for the presence of lymphocytic infiltrates with bile duct destruction and vascular endothelial cell inflammation and invasion. Spleen histology was evaluated for preservation or destruction of lymphoid vesicles. Skin sections were evaluated for lymphocyte infiltration in the subcutaneous area and appendages. Bone marrow sections were evaluated for cellularity and maturation within each major lineage: bone marrow, red blood cells, and megakaryocytes. A → F1 mice developed histological evidence of GVHD in the liver, spleen, bone marrow, and oral mucosa in a blind study. In contrast, recipients of either photochemically treated donor cells (PCT-A) or syngeneic cells (F1) showed no histological abnormalities.

Injected cells varied from 50 nM to 150 μM S-59 concentration and 3 J / c.
Transfusion of a group of mice treated with m2UVA showed complete GV over this range
It showed HD inhibition. This indicates that the dose window in which GVHD can be inhibited by PCT in vivo is similar to that obtained for in vitro assays.

In conclusion of the in vitro and in vivo data, PCT treatment of leukocytes over 3.5 log units of S-59 concentration can inhibit their proliferation in vitro,
And it can inhibit TA-GVHD in vivo. A dose-dependent modulation of leukocyte function in vitro (cytokine synthesis and surface molecule expression) was observed for the same range of concentrations.

Example 6 This example demonstrates that P suitable for suppressing GVHD while preserving the GVL effect of DLI, for application in the treatment of patients with relapsed leukemia after allogeneic BMT, for example.
An approach for determining CT conditions is described. The experimental purpose is summarized below.
A. Determination of the psoralen concentration range to be used in the PCT to prevent lymphocyte proliferation in vitro and thus inhibit GVHD. B. Phenotypic characterization of leukocytes photochemically treated in vivo to determine cell viability and expression of T cell and NK cell surface antigens that are indicative of GVL effects in vivo. C. Qualitative and quantitative analysis of cytokine expression by leukocytes treated in vitro. D. GVH by leukocyte-DLI treated in vivo on mouse leukemia model
Demonstration of inhibition of D.

Photochemical Treatment of Leukocytes with Psoralen A stock solution of psoralen is prepared as illustrated below. 15 meters of AMT
M stock solution was prepared by dissolving 50 mg of AMT powder in 10 mL of distilled water. The solution was mixed vigorously and filtered through a 0.2 μm syringe filter. The concentration of AMT in the filtered solution was adjusted to Shimadzu UV16.
Determined by measuring the absorbance of the solution at 250 nm using a 0U spectrophotometer. The AMT concentration was calculated for the extinction coefficient using the value of 25000 M ^-1 cm ^-1 .

A stock solution of S-59 psoralen was prepared by dissolving S-59 powder in distilled water. The solution was mixed vigorously and filtered through a 0.2 μm syringe filter. The concentration of S-59 in the filtered solution was adjusted to Shimadzu.
Determined by measuring the absorbance of the solution at 250 nm using a UV 160U spectrophotometer. The S-59 concentration was calculated for the extinction coefficient using a value of 25400 M ^-1 cm ^-1 .

8-Methoxypsoralen (8-MOP) is not very soluble in aqueous solution. Therefore, leukocyte preparations treated with 8-MOP and UV light were prepared from 8-MOP-saturated PAS solution (PAS (available from Baxter) from sodium acetate, trisodium citrate, sodium chloride, phosphate buffer). Is an artificial blood substitute (pH = 7.2, 300 mOsm)). 100 mL PA
A saturated solution was prepared by suspending 100 mg 8-MOP in S.
The solution was stirred at room temperature for 16 hours in a dark container. The resulting solution was charged with 0.
Filtered through a 2 μm filter to remove any undissolved 8-MOP. The final 8-MOP concentration was determined using a Shimadzu UV160U spectrophotometer for 2
It was determined by measuring the absorbance at 48 nm. 8-MOP concentration was calculated for the extinction coefficient using the value of 22900 M ^-1 cm ^-1 .

Stock psoralen solution is added to a suspension of purified donor leukocyte aggregates to the desired final concentration. Units treated with 8-MOP were prepared by using PAS saturated with 8-MOP in the preparative procedure discussed above. The purified donor leukocytes are irradiated with UVA light in the photoactivation device to a final dose of 10 ^{−3 to} 100 J / cm ² . The unit was agitated at 70 cycles / min during irradiation. A white blood cell sample is taken prior to irradiation for use as a control.

S-59 is added to the purified leukocyte preparation at a concentration of 10 ^{−4 to} 150 μM. White blood cells are provided at a cell density of 10 to 10 ⁸ cells / mL, preferably 10 ⁷ cells / mL, more preferably 2 × 10 ⁶ cells / mL.

[0229]   A sample of leukocyte aggregates mixed with S-59, 200-450 nm, preferably
First, it is exposed to ultraviolet light having a wavelength of 320 to 400 nm. The cells are preferably about 10 ^-3 ~ 100 J / cm²Receive the amount of light in the range. In a preferred embodiment, the amount of light
Is 3 J / cm²Is. The UV light exposure time is about 1 second to 60 minutes, preferably about 1
Minutes.

Following PCT, leukocyte samples are assayed for their ability to proliferate and participate in leukocyte activity as described above and in the Examples. The following are measured: cell viability and integrity; proliferative activity; surface antigen expression; cytokine secretion; GVHD
And GVL activity.

The following assays can be applied to both alkylator compound treated leukocytes or photochemically treated leukocytes.

A. In Vitro Leukocyte Dose Response Kinetics i) Limiting Dilution Assay (LDA) This assay directly measures the number of cloneable T cells. This number correlates directly with the incidence and severity of GVHD in vivo (Kernan, NA et al., Blood 68: 770-773, 198).
6). LDA sets current guidelines for gamma irradiation of blood products F
Used by DA. This assay is performed by Pelsynthki, M .; Blooo
d 83: 1683-1689, 1994. Briefly, human leukocytes are assayed for the number of proliferating cells after PCT treatment with different concentrations of drug and constant dose of light. Lethal gamma-irradiated pooled allostimulator cells are used to stimulate proliferation. Controls for proliferation include untreated leukocytes (as a positive control for proliferation) and lethal gamma-irradiated pre-PCT leukocytes (negative control). Proliferation is measured by the number of isolated proliferating colonies. The correlation between the dose used and the number of cells whose growth is inhibited is determined for human leukocytes. It is expected that the dose response obtained from this experiment will be similar to that obtained for white blood cells in platelet concentrates.

LDA was performed as follows. The leukocytes are treated with leukocyte apheresis (leuk
was isolated from peripheral blood. RPMI medium that stimulates viable T cells and is supplemented with fetal bovine serum (FBS), phytohemagglutinin (PHA), recombinant interleukin-2 (rIL-2), and T cell growth factor (TCGF). Grow in wells of microtiter plates. Each well was also prepared from a pool of PBMCs from 10 individuals and 500 cG
It contained 10 ⁵ allogeneic stimulator cells irradiated with y-irradiation of y.

[0234]   White blood cells from each untreated control sample were tested in two independent series.
Diluted. 10 in the first series per well^Five10,^Four10,³10,²10, ¹ , And 10⁰Cells, or in the second line 300, 100, 33, 11,
And 100 μL aliquots containing 4 cells were plated. Each dilution,
Plated in 10 replicates. 1.1 x 10⁷Photochemical treatment, including total white blood cells
11 mL aliquots of the prepared samples were cultured and viable T cells were detected. This
100 μL aliquots from the
Started.

Microtiter plates were incubated at 37 ° C. for 3 weeks in a CO ₂ incubator. After 0.5, 1, and 2 weeks of incubation, each well received additional TCGF, FBS, rIL-2, and PHA. Three
At the end of the weekly incubation period, each well was scored for the presence of T cell clones. Wells with one or more T cell clones were scored as positive. Wells without T cell clones were scored as negative. The T cell frequency for each sample was calculated using a least chi-square analysis based on Poisson distribution. The T cell depletion rate was calculated using the following formula: T cell depletion rate = f control / f treatment, where f control and f treatment are T cell frequency for control and treated samples, respectively. .

Control aliquots were untreated or were UVA only or S-5.
Treated with 9 only. One aliquot of γ with a clinical dose of 2500 cGy
Illuminated with a line. To the remaining aliquots, S-59 was added to final concentrations ranging from 10 ⁻⁴ μM to 150 μM. Then, to them, 10 ^{-3 to} 100 Joules /
Irradiation was carried out with an amount of UV light covering a range of cm ² .

Ii) Mixed Leukocyte Response (MLR) Assay This assay, also known as the MLC test, is described in Kraemer, K. et al. H. ,
J. Inv. Derm. 77: 235-239, 1981, and Kraeme.
r, K. H. Et al., J. Inv. Derm. 76: 88-87, 1981. For a review of theory and controls in this assay, see Ba
sic and Clinical Immunology, 8th Edition, Dani
el P.L. States, Abb. Terr and Tristram
G. Parslow (ed.), Appleton & Lange, Norway
k, CT, 1994, 246-247. This assay indirectly measures DNA synthesis induced in lymphocytes by allogeneic stimulation. Two
When lymphocytes of HLA disparate individuals are combined in tissue culture, cells grow, synthesize DNA, and proliferate, while HLA-identical cells remain quiescent. Induced DNA synthesis is determined via incorporation of ³ H-thymidine into nucleic acids inside proliferating cells. Cells are then harvested, washed free of unbound radioactivity and counted in a β counter for internalized radioactivity.

Leukocytes are assayed for their responder and stimulatory functions in a one-way MLR. Standard, appropriate positive and negative controls are included. Response function is assayed by incubating responding cells with lethal y-irradiated pooled human leukocytes (stimulator cells). By lethally γ-irradiating treated leukocytes (treated with PCT or other alkylator compounds) to block thymidine uptake and then incubating their stimulatory functions with normal pooled human leukocytes. decide. The results obtained as a function of processing dose are compared with a two-way MLR.

MLRs provide a subset of the information provided by LDA, but may provide information about the ability of treated leukocytes to stimulate other cells. Stimulation of treated leukocytes in the MLR is used as a model for the induction of an immune response against leukemia and can be activated by DLI in BMT infused patients. In that case, an immune response against leukemia (GVL effect) may be initiated by host cells or donor cells present in BMT.

B. Phenotypic Characterization of Treated Leukocytes i) Phenotypic Antigenic Marker Expression The effect of PCT or alkylator treatment on the expression of phenotypic antigenic markers was determined by the use of an appropriate fluorescently labeled antibody (Pharmingen). , And standard FACS-SCAN analysis. CD2, an antigenic marker known to be involved in interactions associated with T cell and NK cell activation and immune function
, CD28, CTLA4, CD40 ligand (gp39), CD18, CD25
, CD69, and CD16 / CD56, the presence of the appropriate fluorescently labeled antibody (
Pharmingen) and Coligan, J .; E. Curr
ent Protocols in Immunology: Current
Protocols. New York, John Wiley & Son
s, Inc. , 1991 as a function of process dose. The stability of surface molecules under treatment conditions is not expected to be affected given the mild nature of PCT and the associated treatment to proteins; however, their induction of expression is dose-dependent. Is predicted.

See Example 14 for characterization of treated human leukocytes with respect to proliferation, surface marker expression, cytokine synthesis, and cytotoxic activity.

Ii) Leukemia cell line lysis To assay for direct GVL effects induced by treated cells,
Cytolytic potency is tested against a range of human and mouse leukemia cell lines (available from ATCC). The experiment was performed by Jiang, Y. et al. Z. Et al, Bone Marr
ow Transplant 8: 233-238, 1991. Leukemia cells were cultured under standard conditions in Coligan, J. et al. E. Curr
ent Protocols in Immunology: Current
Protocols, John Wiley & Sons, Inc. , New York, 1991 and labeled with ⁵¹ Cr. Any lysis achieved is measured by the release of ⁵¹ Cr radioactivity in the supernatant and compared to controls for background and total lysis. The effect of treatment dose on the cytolytic capacity of treated T cells is monitored. Experiments are performed by adding increasing amounts of treated leukocytes to see if lysis occurs. The experiment is T
It can be repeated with a subset of cells. Direct lysis of leukemic strains provides a direct mechanism for GVL effects, but its lack may imply an indirect mechanism for induction of GVL effects by DLI. Therefore, cytotoxic activity can instead be determined by measuring cytokine production, eg, IFN-γ, IL-2, or GM-CSF production.

Iii) Viability of cells in vivo To assess the viability of treated cells in vivo, after treatment with various doses,
Male mouse leukocytes are injected into female syngeneic or allogeneic recipients. The PCR procedure (Goodarzi, MO et al., Transfu) where the presence of treated cells in the circulation was applied to blood samples taken at desired time points after splenocyte injection.
sion 35: 145-149, 1995). This procedure detects the presence of Y chromosome-specific gene sequences that are only present in male (donor) cells, and has a sensitivity of 1-5 cells per 50 μL of host blood. This experiment determines the effect on the elimination of injected cells in living animals, as well as the relationship between treatment dose and cell circulation in vivo. Viability of treated cells in vivo was also determined by Johnson et al., Scand. J. Immunol. 45: 511 ~
514, 1997, and can be monitored by PKH-26 labeling of leukocytes prior to infusion.

C. Quantitative and Qualitative Characterization of Cytokine Synthesis Quantitative and qualitative cytokine measurements were performed using the commercially available Elisa kit (R & D System) in culture supernatants after co-culture of effector cells with stimulator cells.
s). The assay is performed according to the instructions provided by the Elisa manufacturer. Results are evaluated by comparing the absorbance measurements for each sample to a standard curve generated by the cytokine standards supplied with each assay kit. Cytokine measurements are made on treated leukocytes as a function of compound concentration and, in the case of PCT, light intensity and time interval between treatment and measurement. IL-1, IL-2, IL-4, IL-10, IFN-γ cytokines are measured for T cell activation and an established role in GVHD / GVL.

The measurement of cytokine synthesis by treated leukocytes is important in determining the ability of cells to function as inducers of the immune response via their production; it also
Acts as a measure of biochemical function and protein synthesis.

See Example 14 for characterization of treated human lymphocytes with respect to proliferation, cell surface expression, cytokine synthesis, and cytotoxic activity.

D. Use of In Vivo Mouse Models Demonstrating Inhibition of GVHD and Induction of GVL Effects Mice are well established models for studying graft versus host disease and graft versus leukemic graft effects. Is. To differentially evaluate the effects of treated leukocytes on GVHD and GVL, Johnson, B. et al. D. Et al, Bone Marr
ow Transplant, 11: 329-336, 1993, and Joh.
Nson, B.N. D. Et al., Blood 85: 3302-3312, 1995 using the transplanted mouse model. In this model, female AKR
/ J mice (H-2 ^k ) were irradiated to pre-lethal dose (9 Gy), then MHC matched female B10. It gave BR donor (H-2 ^k) derived from 10 ⁷ BM cells and 3 × 10 ⁷ spleen cells. This treatment has been shown to result in acute GVHD clinical symptoms and death within 50 days. Removal of splenocytes from the infusion results in complete survival of mixed chimeras, whereas BMT does not result in 50% mortality. In this example, mouse splenocytes were infused to test the inhibition of GVHD by treatment, as measured by mortality, and also to provide the lowest dose to inhibit the development of GVHD. Used for previous processing. Splenocytes are the accepted replacement for blood leukocytes in mice, as the blood volume in mice is so small for such experiments.

To assess GVL reactivity of treated cells, recipients are injected with PA allogeneic splenocytes, for example, 21 days after BMT, then challenged with AKR leukemia cells 7 days later. Injection of 5 × 10 ⁴ AKR leukemia cells into mice after recovery from BMT (28 days) is known to result in leukemia and 95% mortality 25 days after leukemia challenge (Johnson, BD). .., Transplan
station 54: 104-112, 1992). On the other hand, injection of allogeneic splenocytes 21 days after BMT showed GVL against challenge with AKR cells 7 days later.
It is known to induce an effect. This course of action results in 70% survival after 70 days. If prevention of GVHD has been demonstrated, treatment of splenocytes prior to infusion should
Allows detection of induction of GVL in the absence of VHD. It is measured by the viability of treated splenocyte injected BMT recipients (challenge with AKR cells).

The effect of DLI using treated leukocytes on the survival of leukemia challenge is tested as a function of treatment dose and compared to a control group in which an injection of untreated splenocytes is performed. Surviving mice are described in Johnson, B .; D. Bon, Bon
Chimerism and leukemia burden are tested through the use of a reported PCR assay as described in e. Marrow Transplant, 11: 329-336, 1993.

In this experimental model, the cause of death due to leukemia can be diagnosed either by white blood cell count immediately before death or by PCR analysis of blood. Because leukemia is AK
This is because it causes the expansion of R leukemic cells. Primers specific for AKR leukemia cell-specific markers are used for PCR analysis. Mortality from GVHD is diagnosed by low white blood cell count, and splenic atrophy, as well as characteristic clinical symptoms (changes in soft skin and posture, sticky feces, etc.). The efficacy of PA-DLI against minimal residual disease or higher tumor burden is tested by injecting smaller or larger numbers of AKR leukemia cells.

Leukocyte treatment of the invention, eg, by the PCT approach, produces a statistically significant increase in survival of treated cells with DLI-treated mice after a leukemic challenge is administered under conditions that do not cause GVHD. When induced, it is considered to be efficient in inducing the GVL effect. The genotype of the surviving animal should also be fully chimeric. Because it is associated with a higher incidence of disease-free long-term survival in humans. Increased survival is easy to demonstrate within 100 days after the original BMT.

[0252]   Splenocytes are subjected to treatment of donor leukocytes under the conditions described above.

See Example 15 for the provision of GVL effects in the absence of GVHD by treated leukemia in a mouse model system.

Examples 7-12 The following specific examples are relevant data for compounds useful to practitioners to illustrate preparative methods for representative alkylated compounds useful in the methods of the invention. Are provided to illustrate, and to illustrate the manner in which the effect of a compound is determined. All NMR spectra were measured in CDCL ₃ unless otherwise stated.
n 200MHz instrument recorded; chemical shifts tetramethylsilane (TMS
) Is the standard. IR spectrum is Perkin Elmer FTI
Recorded using R. HPLC, using a YMC C8 column was performed with a gradient mode using 5 mM CH ₃ CN as 5 mM H ₃ PO ₄ aqueous solution and mobile phase B as mobile phase A. Samples were prepared in DMSO or ethanol and kept below 15 ° C before injection.

[0255]   Table 2 shows the nomenclature of compound numbers used for various compounds.

[0256]

[Table 2]

Example 7 Synthesis of β-alanine, N- (2-carbomethoxyacridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride (Compound IV) Step A ． β-alanine, N- (tert-butoxycarbonyl), 2- [bis (2-hydroxyethyl) amino] ethyl ester N- (tert-butoxycarbonyl) -β in dry THF (200 mL).
-Alanine (20.3 g, 107 mmol) and 4-methylmorpholine (1
3.0 mL, 12.0 g, 119 mmol) in a stirred solution at -15 <0> C under nitrogen isobutyl chloroformate (13.9 mL, 14.6 g, 10).
7 mmol) was added resulting in the instantaneous formation of a white precipitate (4-methylmorpholine.HCl). The reaction mixture was stirred at -15 ° C for 5 minutes, followed by -15 ° C.
Triethanolamine (48.3 g, 324 in dry THF (150 mL) at
The reaction mixture was transferred to a flask containing a stirred solution of (mmol). The reaction mixture was warmed to 23 ° C. and stirred for an additional 1.5 hours, followed by removal of the precipitate by vacuum filtration. The THF was then removed from the filtrate under vacuum and the remaining viscous yellow oil was partitioned between water (500 mL) and EtOAc (5 x 150 mL). The combined organic layers were dried over Na ₂ SO ₄ . The solvent was removed under vacuum to give 25.8 g (75%) of the desired product, β-alanine, N- (tert-butoxycarbonyl), 2- [bis (2-hydroxyethyl) amino] ethyl ester. Obtained as a pale-yellow oil.

[0258]

[Equation 1]

Step B. β-alanine, N- (tert-butoxycarbonyl), 2- [bis (2-tert-butyldimethylsilyloxyethyl) amino] ethyl ester β-alanine, N- (from step A in acetonitrile (70 mL). ter
t-butoxycarbonyl), 2- [bis (2-hydroxyethyl) amino] ethyl ester (22.7 g, 70.9 mmol) and imidazole (11.1 g).
, 163 mmol) was cooled to 0 ° C. under nitrogen. tert-Butylmethylsilyl chloride (534 mg, 3.54 mmol) was then added and the reaction mixture was stirred at 0 ° C. for a further 5 minutes. The reaction mixture was warmed to 23 ° C. and stirred for 2 hours, followed by removal of white precipitate (imidazole.HCL) obtained by vacuum filtration. The acetonitrile was removed from the filtrate under vacuum and the resulting material was partitioned between saturated brine (600 mL) and EtOAc (3 x 200 mL). The combined organic layers were dried over Na ₂ SO ₄ . The solvent was removed under vacuum to give 35.2 g (90%) of the desired product, β-alanine, N- (ter.
t-Butoxycarbonyl), 2- [bis (2-tert-butyldimethylsilyloxyethyl) amino] ethyl ester was obtained as a yellow oil.

[0260]

[Equation 2]

Step C β-Alanine, 2- [bis (2-tert-butyldimethylsilyloxyethyl) amino] ethyl ester β-Alanine, N- (tert-butoxycarbonyl), 2- [from Step B
Trifluoroacetic acid (5 mL) was added to a flask containing bis (2-tert-butyldimethylsilyloxyethyl) amino] ethyl ester (3.01 g, 5.48 mmol) without a solvent, and CO ₂ gas was generated. The reaction mixture was stirred for 5 minutes and trifluoroacetic acid was removed under vacuum. The rest of the material is saturated with NaH
Partitioned between CO ₃ (100 mL) and EtOAc (3 × 30 mL). The combined organic layers were dried over Na ₂ SO ₄ . The solvent was removed under vacuum and 2.45 g (
100%) of the desired product, β-alanine, 2- [bis (2-tert-butyldimethylsilyloxyethyl) amino] ethyl ester was obtained as a pale yellow oil.

[0262]

[Equation 3]

Step D. β-alanine, N- (2-carbomethoxyacridin-9-yl),
2- [bis (2-hydroxyethyl) amino] 12.5 hours in CHCl ₃ ethyl ester 10 mL, at room temperature, beta-alanine, 2- [bis (2-tert-butyldimethylsilyloxy ethyl) amino] The ethyl ester (736 mg, 1.64 mmol) was reacted with methyl 9-methoxyacridine-2-carboxylate (669 mg, 2.50 mmol). Then, the precipitate (acridone) was filtered off, and the filtrate was washed with saturated NaHCO ₃ aqueous solution (100 mL) and C
Partitioned with HCl ₃ (3 × 35 mL). The combined organic layer is Na ₂ SO 4
Dried in vacuo and concentrated in vacuo to give 1.61 g of a viscous brown oil. Deprotection of the resulting diol was carried out by dissolving this crude intermediate in 3.0 mL of THF under nitrogen and cooling to 0 ° C. and treating with HF / pyridine (1.0 mL). It was The solution was warmed to room temperature with stirring for 1 hour. Volatiles were removed under vacuum and the residue was saturated aqueous NaHCO ₃ (100 mL) and CHC.
Partitioned with L ₃ (3 × 35 mL). The combined organic layers were dried and concentrated to give 649 mg of a brown-yellow solid. Preparative TLC (C-18, CH ₃ C
N) is the desired diol, β-alanine, N- (2-carbomethoxyacridin-9-yl), 2- [bis (2-hydroxyethyl) amino] ethyl ester (80% by HPLC) in 20% yield. % Purity).

[0264]

[Equation 4]

Step E. β-alanine, N- (2-carbomethoxyacridin-9-yl)
, 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride of β-alanine, N- (2-carbomethoxyacridin-9-yl), 2- [bis (2-hydroxyethyl) amino] ethyl ester Conversion to the dichloro compound was accomplished by a method similar to that of Peck et al. (J. Am. Chem. Soc. 1959, 81: 3984). Step D in solvent-free SOCl ₂ (6 mL)
A yellow solution of the product from (41 mg, 0.090 mmol) was stirred for 20 hours at room temperature. The SOCl ₂ was then removed under vacuum to give a yellow solid (dihydrochloride salt). This material was then treated with saturated NaHCO ₃ (50 mL) and CH ₂ Cl ₂ (3
X 20 mL). The combined organic layers were dried over Na ₂ SO ₄ .
The solvent was removed under vacuum to give 35.4 mg of the dichloro compound free base as an orange gum.

[0266]

[Equation 5]

No other carbon was observed. The HCl salt was precipitated from CH ₂ Cl ₂ by adding 1M HCl in ether to give β-alanine, N- (2-carbomethoxyacridin-9-yl), 2- [bis (2-chloroethyl). Amino] ethyl ester dihydrochloride (Compound IV) was obtained as a yellow solid (81% pure by HPLC). β-alanine, N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride (Compound V) was prepared in a similar manner. Therefore, using 9-methoxyacridine instead of methyl 9-methoxyacridine-2-carboxylate in Step D, the intermediate diol was replaced with a yellow oil (H
Obtained by PLC as 74% purity) (7.1%).

[0268]

[Equation 6]

Intermediate diol (37.3 mg, 0.079) in thionyl chloride (4.0 mL).
The solution of 3 mmol) was stirred at 23 ° C. for 7.5 hours. Thionyl chloride was removed under vacuum to give a yellow oil. This material was dissolved in ethanol (-4 mL) and the solvent removed under vacuum. This material was then dissolved in CH ₂ Cl ₂ (4 mL),
The solvent was then removed under vacuum and this process was repeated twice. This material was then triturated with hexane (3 x 4 mL) to give 40.0 mg (42% pure by HPLC) of the product in the form of a yellow, hygroscopic glassy solid. A small amount of this material was added to saturated NaHCO ₃ for analysis.
And CH ₂ Cl ₂ and subsequently the combined organic layers were converted to the free amine by drying over Na ₂ SO ₄ and removing the solvent under vacuum.

[0270]

[Equation 7]

Following the procedure described above, but replacing N- (tert-butoxycarbonyl) -β-alanine with N- (tert-butoxycarbonyl) -4-aminobutyric acid, 4-aminobutyric acid N-[(2- Carbomethoxyacridin-9-yl), 2-
[Bis (2-chloroethyl) amino] ethyl ester dihydrochloride, compound VI (H
Led to the preparation of 78% purity by PLC).

[0272]

[Equation 8]

Example 8 Triethanolamine in Example 7, Step A was treated with 3- [N, N-bis (2-te).
rt-Butyldimethylsilyloxyethyl)] aminopropanol,
Then by continuing from step C, β-alanine, N- (2-carbomethoxy-acridin-9-yl), 3- [bis (2-chloroethyl) amino] propyl ester dihydrochloride, compound VIII, (HPLC Led to a preparation of 63% purity).

[0274]

[Equation 9]

Example 9 The compound synthesized in Example 7 can also be prepared by the following method: β-
Alanine, N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride (Compound V): Method II Step A: β-alanine, N- (acridin-9-yl) ), Methyl ester hydrochloride 9-chloroacridine (11.7 g, Organic Synthesis,
Coll. Vol III, page 57), β-alanine methyl ester hydrochloride (9
． 9 g) and sodium methoxide (3.26 g) combined, 60 mL
Of methanol was added. Stir this mixture with a magnetic stirrer, 5.
Refluxed for 5 hours. The heat was removed and the suspension was filtered while warm (<35 ° C). The solid salt was rinsed with an additional 10 mL of methanol and the combined dark green filtrate was concentrated to give 21 g of a wet green-yellow solid.

This solid was dissolved in 350 mL of boiling 2-propanol and crystallized at room temperature. The obtained crystals were mixed with about 15 mL of 2-propanol and 15 m.
Rinsed with L hexane, then dried in air, 15.5 g bright yellow product,
β-alanine, N- (acridin-9-yl), methyl ester hydrochloride (yield 7
8.5%) was obtained.

[0277]

[Equation 10]

Step B: β-alanine, N- (acridin-9-yl), 2- [bis (2-hydroxyethyl) amino] ethyl ester dihydrochloride β-alanine, N- (acridin- 9-yl), methyl ester hydrochloride (5.00 g) in toluene (750 mL), saturated aqueous Na ₂ CO ₃ solution (20
Partitioned between 0 mL) and H ₂ O (50 mL). This water layer is again converted to toluene (
3 × 250 mL) and combine the organic layers and sat. Na ₂ C
It was washed with an O ₃ aqueous solution (50 mL). The volume of toluene was reduced to approximately 100 mL by rotary evaporation. Then, triethanolamine (3
0 mL) was added to form a partially immiscible system. Then M
A solution of NaOMe (50 mg) in OH (2 mL) was added. The solvent was rapidly removed from the reaction mixture by rotary evaporation with shaking at room temperature. After removing the solvent, the reaction mixture was left under vacuum for an additional 1 to 1.5 hours to obtain a syrupy solution.

[0279]   CH this crude mixture₂Cl₂Minutes between (200 mL) and saline (200 mL)
Then, excess triethanolamine was removed. CH saline layer₂Cl₂(5 x 1
(00 mL). Combine the organic layers and wash with brine (50 mL)
And then extracted with 0.5M HCl (2 × 100 mL). Acid aqueous layer
Mating and CH₂Cl₂Wash with (50 mL). This aqueous acid solution is converted into CH ₂ Cl₂Powdered K in the presence of (200 mL)₂CO₃Basic (using solid)
Turned into The organic layer is separated and the aqueous layer is re-CH₂Cl₂(5 x 100 mL)
It was extracted with. The combined organic layers were washed with brine (50 mL) and dried over anhydrous Na₂S
O_FourDried (solid) and stripped of solvent to give a sticky yellow gummy crude diol
Free amine (5.02 g) was obtained. This material is an alternative method by NMR.
Therefore, it was the same as that prepared in Example 1.

A portion of the above crude product (0.400 g) was vigorously stirred with isopropanol (100 mL) and acidified with 1M HCl in ether. The slurry was cooled and the first precipitate was discarded. As a second crystal after half removal of the solvent, a bright yellow crystalline solid (0.200 g) with a purity higher than 95% by HPLC.
Β-alanine, N- (acridin-9-yl), 2- [bis (2-hydroxyethyl) amino] ethyl ester dihydrochloride was obtained.

[0281]

[Equation 11]

Step C: β-alanine, N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride from Step B in CH ₃ CN (0.5 mL). To a stirred suspension of β-alanine, N- (acridin-9-yl), 2- [bis (2-hydroxyethyl) amino] ethyl ester dihydrochloride (113 mg, 0.24 mmol) in SOCl ₂ (
0.5 mL) was added. The resulting yellow solution was stirred at 23 ° C. for 16 hours, followed by removal of volatiles under vacuum. The residual orange oil was dissolved in EtOH (~ 2 mL) and the EtOH was removed under vacuum to give a yellow solid. This material was then triturated with hexane (2 x 3 mL). Removal of the remaining solvent under vacuum gave 123 mg of the desired material, β-alanine, N- (acridine-
9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride, (93% pure by HPLC) was obtained as a yellow solid.

[0283]

[Equation 12]

Example 10 β-alanine, N- (4-methoxy-acridin-9-yl), 2- [bis (
2-Chloroethyl) amino] ethyl ester dihydrochloride, compound IX.

Β-alanine, N- (4-methoxy-acridin-9-yl), methyl ester was 1.4 g (5.84 mmol) of 4,9-dimethoxyacridine, 0.8.
9 g (6.42 mmol) β-alanine methyl ester hydrochloride and 20 mL
Of methanol and then heated to reflux under nitrogen for 12 hours to prepare. The reaction was then concentrated under vacuum and CHCl ₃ -isopropanol (
50mL, 4: 1v / v) was dissolved in, _{and, 50% NH 4 OH (2} × 25m
L) and brine (1 x 25 mL). The organic layer was dried over Na ₂ SO ₄ and concentrated under vacuum to give 1.24 g (68%) of its methyl ester (more than 74% pure by HPLC) as a yellow oil;

[0286]

[Equation 13]

This was converted to the diol under the conditions described in Example 3, Step B to give 647 m
g of yellow oil was obtained. HPLC analysis of this crude product showed a yield of 85% (λ = 2
78 nm).

[0288]

[Equation 14]

This was treated with thionyl chloride as described in Example 3, Step C, to give β-
Converted to alanine, N- (4-methoxy-acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride. The crude product was flash filtered (SiC) with ethyl acetate followed by 10% methanol-ethyl acetate.
O ₂ ) and, via apparent decomposition of some of the product on the column, gave 58 mg of a yellow oil.

[0290]

[Equation 15]

The reaction is concentrated under vacuum to azeotropically remove excess thionyl chloride (2 × 5 m).
The dihydrochloride salt could be isolated in crude form by L. HPLC
The analysis showed that the starting material was completely consumed and that 4-methoxyacridone (Rγ = 2
2.3 min) was the main impurity.

[0292]

[Equation 16]

Similarly, from 3-chloro-9-methoxy-4-methylacridine to β-alanine, N- (3-chloro-4-methylacridin-9-yl), 2- [bis (2-
Chloroethyl) amino] ethyl ester dihydrochloride, compound X, was prepared.

[0294]

[Equation 17]

Similarly, from 6-chloro-2,9-dimethoxyacridine to β-alanine, N
-(6-Chloro-2-methoxyacridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester dihydrochloride, Compound XIII, was prepared.

[0296]

[Equation 18]

Example 11 β-Alanine, [N, N-bis (2-chloroethyl), 3-[(6-chloro-
2-Methoxyacridin-9-yl) amino] propyl ester dihydrochloride, compound XI.

Step A β-alanine, [N, N-bis (2-triisopropylsilyloxy) ethyl] ethyl) ester β-alanine ethyl ester hydrochloride (1.
99 g, 12.9 mmol), was _{K 2 CO 3 (6.0g, 43.4mmol} ) and ethyl iodide propyl silyl ether (9.47 g, the slurry 28.9 mmol) was refluxed for 5-7 days. After vacuum evaporation of the solvent, the solid was triturated with CH ₂ Cl ₂ . The organic layer was washed with dilute aqueous Na ₂ CO ₃ solution, then brine and dried over anhydrous Na ₂ SO ₄ . The crude product was purified by silica gel chromatography (1: 4 EtOAc / hexane) to give 5.60 g of oily β-alanine, [N, N-bis (2-triisopropylsilyloxy) ethyl] ethyl).
The ester, (83.1%) was obtained.

[0299]

[Formula 19]

Step B β-alanine, [N, N-bis (2-triisopropylsilyloxy) ethyl] -β-alanine from Step A above, [N, N-bis (2-triisopropylsilyloxy)] Ethyl] ethyl) ester (5.60 g, 10.8 mmol) and lithium hydroxide (0.59 g, 14.1 mmol) were stirred in ethanol,
Refluxed for 3 hours. The solvent was removed and the composition was treated with CH ₂ Cl ₂ and dilute NaHC.
Partitioned with aqueous O ₃ solution. The organic layer was washed with brine, dried over anhydrous Na2SO4, and the solvent was removed and β-alanine, [N, N-bis (2-triisopropylsilyloxy) ethyl]-was pale yellow oil (5. 03 g, 95.1%).

[0301]

[Equation 20]

[0302]   Step C β-alanine, [N, N-bis (2-hydroxyethyl)], 3- [
(6-Chloro-2-methoxyacridin-9-yl) amino] propyl ester   CH₂Cl₂In (1 mL), β-alanine from step B above, [N, N-bi
(2-triisopropylsilyloxy) ethyl]-(51.0 mg, 0.10
4 mmol) was stirred under nitrogen. When cooling on an ice bath, SOCl2 (0.5
mL) was added dropwise and the reaction was stirred for 2.25 hours. Excess SOCl ₂ Is removed from the reaction mixture and then dried CH₂Cl₂(0.5 mL) and add
The solution was then cooled in an ice bath under nitrogen. CH₂Cl₂9- (3 in (1 mL)
-Hydroxy) propylamino-6-chloro-2-methoxy-acridine (29
． 0 mg, 91.5 mmol) of cooled slurry was added. 0.5 hours later,
CH this mixture₂Cl₂And NaHCO₃Partitioned with aqueous solution. This organic layer
Wash with brine, anhydrous Na₂SO_FourAfter drying, the solvent was removed. The obtained gum is
Triturate with hexanes and remove the solvent from the hexane extract to
A very crude mixture of the starting material and the product protected with liisopropylsilyl (
53.5 mg) was obtained.

To remove the triisopropylsilyl group, a portion of the crude protected diol (33.
1 mg) was stirred in ice-cold THF (1 mL). HF / pyridine (0.
After addition of 5 mL), the mixture was stirred for 2.5 h under a balloon filled with nitrogen at ambient temperature. The reaction mixture was partitioned between CH ₂ Cl ₂ and aqueous NaHCO ₃ and the organic layer was washed several times with dilute aqueous NaHCO ₃ to remove excess HF / pyridine. After pre-drying with brine and then with anhydrous Na ₂ SO ₄ , the solvent was removed to give crude diol (13.1 mg).

This was combined with additional crude diol (5.0 mg) and 95 CH ₂ C
and purified by C-18 preparative TLC to the l ₂ / 5iPA / 1TFA as eluent. After partitioning the salt between CH ₂ Cl ₂ and aqueous NaHCO ₃ , the organic layer is dried over brine, then anhydrous Na ₂ SO ₄ and the solvent removed to remove the free base of the diol, β. -Alanine, [N, N-bis (2-hydroxyethyl)], 3-
[(6-Chloro-2-methoxyacridin-9-yl) amino] propyl ester, (5.0 mg) was obtained.

[0305]

[Equation 21]

Step D β-alanine, [N, N-bis (2-chloroethyl)], 3-[(6
-Chloro-2-methoxyacridin-9-yl) amino] propyl ester dihydrochloride, compound XI.

Β-alanine, [N, N-bis (2-hydroxyethyl)], 3-from above
Dissolve [(6-chloro-2-methoxyacridin-9-yl) amino] propyl ester (4.0 mg, 0.0073 mmol) in CH ₂ Cl ₂ (1 mL),
Then cooled in an ice / water bath. SOCl ₂ (0.1 mL) cooled with ice was added and the reaction was stirred for 4 hours at room temperature. The reaction mixture was stripped of solvent, triturated with hexane and partitioned between CH ₂ Cl ₂ and aqueous NaHCO ₃ . The organic layer is dried over brine, then anhydrous Na ₂ SO ₄ , and
The solvent was removed to give the dichloro compound as a yellow gum.

[0308]

[Equation 22]

The free amine was stirred in chilled CH ₂ Cl ₂ , acidified with 1M HCl in ether and the solvent removed with a few drops of methanol to give the desired compound, β-alanine, [N. , N-bis (2-chloroethyl)], 3-[(6-chloro-2-methoxyacridin-9-yl) amino] propyl ester dihydrochloride (2
． 5 mg), (3.5 mg, 81%) was obtained as a yellow solid.

Other than using 6-chloro-9- (2-hydroxy) ethylamino-2-methoxy-acridine instead of 6-chloro-9- (3-hydroxy) propylamino-2-methoxy-acridine. Prepared a similar diol in a similar manner as provided in Step C above.

[0311]

[Equation 23]

This was treated with β-alanine, [N, N-bis (2-chloroethyl)], 2-[(6-chloro-2-methoxyacridin-9-yl) amino] by a method similar to Step D. Converted to ethyl ester dihydrochloride, compound XII.

[0313]

[Equation 24]

Example 12 [N, N-Bis (2-chloroethyl)]-2-aminoethyl 4,5 ', 8-trimethyl-4'-psoralen acetate hydrochloride, Compound XIV Step A: [N, N-bis (2-hydroxyethyl)]-2-aminoethyl 4,
5 ', 8-Trimethyl-4'-psoralen acetate Methyl 4,5'8-trimethyl-4'-psoralen acetate (250 mg, 0.83
2 mmol), triethanolamine (12 mL) and 1 MH in ether.
A slurry of Cl (2 mL) was stirred at 100 ° C. for 2 hours. The resulting clear brown solution was cooled to room temperature, and partitioned between CH ₂ Cl ₂ and saturated aqueous NaHCO _3. The organic layer was rinsed several times with saturated aqueous NaHCO ₃ . Anhydrous Na ₂ S
After drying over O ₄ , the solvent was removed under vacuum and the residue was diluted with CH ₂ Cl ₂ and 1M.
Partition with aqueous HCl. The aqueous layer was rinsed several times with CH ₂ Cl ₂ and then made basic with K ₂ CO ₃ (solid) in the presence of organic solvent. The organic layer containing the neutral product was washed several times with water, then dried and concentrated. Repeating this acid-base extraction procedure gave the desired product as a beige solid (84.3).
mg, 24.3%).

[0315]

[Equation 25]

Step B: [N, N-bis (2-chloroethyl)]-2-aminoethyl 4,5 '
, 8-trimethyl-4'-acetate psoralen hydrochloride CH ₂ Cl ₂ (1mL) above diol in (9.8 mg, 0.023 mmol
Thionyl chloride (0.2 mL) was added to the ice bath mixture of 1) and stirred under nitrogen overnight at room temperature. The resulting slurry was concentrated and then triturated with hexanes to give the desired product (6.2 mg, 53.9%) as a yellowish white solid.

[0317]

[Equation 26]

Example 13: Illustrative BMT Light or Mini Transplant Protocols Patients with CLL are non-dissected (n) prior to transplant with allogeneic peripheral blood stem cells.
onlabive) Pre-curing is given. After transplantation, support with DLI as needed to achieve maximal graft-versus-malignant effect. Mild pre-curing is based on fludarabine (FAMP) and modified according to malignant type. For advanced and previously treated CLL, patients receive 300 mg / m2 / dx3 FAMP with 30 mg / m2 / dx3 cyclophosphamide. Richter and large cell lymphoma (LCL) were FAMP 30 mg / m2 / dx2, cisplatin 25 mg / m2 / d / CIx4, and Ara-c 0.5 gm mg.
Treat with a regimen consisting of / m2 / dx2. GV with the exception of treatment for LCL
Do not give HD prevention. This regimen is intended to create a chimerism and allow transplantation while reducing the toxicity of conventional induction therapy. DLI may be given one month post transplant to boost the transplant. Successful transplants enhance the graft-versus-leukemia effect,
It then allows subsequent DLI to be administered as needed to promote immune recovery.

Example 14 In Vitro Studies with Human Peripheral Blood Lymphocytes Human Peripheral Blood Lymphocytes (PBL) that have been subjected to PCT and cultured in vitro to characterize the particular type of treated cell population. I examined some characteristics of. These included viability, T cell proliferation, cytokine synthesis and secretion, surface antigen expression, in vitro cytotoxicity, and Fas ligand expression.

Viability PBLs subjected to 10 nM S-59 at 3 J / cm ² UVA were assayed for viability by trypan blue exclusion. Survival is 75% after 2 days, 3
50% after day and 25% after 4 days. In a related experiment, dose-dependent survival of treated lymphocytes was observed.

Proliferation Lymphocyte proliferation was measured by ³ H thymidine incorporation into DNA of allogeneic stimulated T cells. Peripheral blood mononuclear cells (PBMC) were isolated from 3 donors, pooled, gamma-irradiated (2500 cGy) to prevent autostimulation, and used as stimulator cells in MLR. PBMCs isolated from a fourth donor were used as responder cells. Responder cells were treated with 3 J / cm ² UVA at 0.05, 0.5, or 5 nM 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen ("S-59. )) And co-cultured with stimulator cells. Control untreated responder cells were also co-cultured with stimulator cells. After 6 days of culture, ³ H
Thymidine was added to the cultures and one day later cells were harvested and cell samples were analyzed by scintillation counting to determine ³ H thymidine incorporation.
FIG. 8 shows that proliferation of treated leukocytes, measured by ³ H thymidine incorporation in MLR, was eliminated in a dose-dependent manner between S-59 of 0.1 nM and 10 nM, with complete inhibition of growth of 10 nM S. It shows that it occurred at -59.

The proliferation potential of treated leukocytes was also analyzed by another method. Here, treated cells were activated by surface-bound anti-CD3 antibody. In this assay, PBMC
Is either untreated or 0.0001, 0.001 or 0.01 μM S-59
At a UVA dose of 3 J / cm ² or irradiation in the absence of S-59. The cells were then incubated in plates with anti-CD3 antibody attached at 37 ° C. in 5% CO ₂ to induce polyclonal activation of T cells and ³ H thymidine incorporation in culture 1. It was measured after 2 and 3 days. The results (FIG. 9) show a dose-dependent decrease in growth potency under all treatment conditions and complete growth inhibition with 10 nM S-59.

Cytokine production Human lymphocytes were cultured in MLR as described in the previous section on proliferation analysis. Responder cells were treated with 3.0 J / cm ² UVA at different concentrations of S-59 (0
． 05, 0.5, or 5 nM). Samples of MLR cell culture medium were taken after 2, 4, 6 and 7 days of culture. These samples were tested for IL-2 and IFN-γ levels by sandwich enzyme-linked immunosorbent assay and the results were quantified spectrophotometrically. The standard solution was used for the construction of a standard curve for concentration (pg / ml) and absorbance, from which the cytokine concentration in the test samples was determined.

An analysis of IL-2 production is shown in Figure 10A. I by untreated lymphocytes and lymphocytes treated with lower concentrations of S-59 (ie 0.05 and 0.5 nM)
L-2 secretion peaked at day 2 of culture and then decreased due to IL-2 consumption by expanding lymphocytes in the population. In contrast, 5 nM S-5
In lymphocytes treated with 9, IL-2 was not consumed by non-proliferating leukocytes and 4
At days 6, 6 and 7, it remained at a much higher level in the medium.

IFN-γ production by stimulated PBMC was unaffected or somewhat increased by treatment with S-59 + UVA. See FIG. 10B. The combined results of the IL-2 and IFN-γ analyzes indicate that under conditions where proliferation is inhibited, treated cells can nonetheless synthesize and secrete cytokines associated with T cell activation.

Surface Antigen Expression Analysis of surface marker expression on treated lymphocytes was analyzed. Lymphocytes, Fico
Obtained by 11 gradient centrifugation and activated by in vitro stimulation with surface-bound anti-CD3 antibody. Expression of surface antigens by activated treated lymphocytes on CD
It was measured by fluorescence activated cell sorting (FACS) analysis using fluorescent antibodies against 69, CD25 (IL-2 receptor), or CD40L. Expression was measured as a function of time after activation and compared to expression by untreated cells.

CD69 is an early marker of lymphocyte activation. FIG. 11A shows that cells treated with either 1 nM or 10 nM S-59 + 3 J / cm ² UVA have similar or slightly higher surface CD69 levels up to 48 hours post treatment than untreated cells. Thus, treated cells are unable to proliferate, but activation-related signaling pathways remain functional.

CD25 is the IL-2 receptor and its expression is detected somewhat later (after activation) than CD69. FIG. 11B shows that in cells treated with either 1 nM or 10 nM S-59 + 3 J / cm ² UVA, surface CD25 levels are essentially unaffected compared to untreated cells.

The CD40 ligand (CD40L) on T cells recognizes the CD40 molecule expressed on the surface of B cells as part of the T cell activation process. C on T cell
Inhibition of D40L expression can induce cell unresponsiveness or anergy. Therefore, stable expression of CD40L on T cells after treatment is an indicator of normal T cell activation. The effect of PCT on the expression of CD40L was examined after activation of treated cells with anti-CD3 antibody (Fig. 12). The results of this analysis show that for treatment conditions that are subsequently deprived of proliferative capacity, CD40L expression by treated cells closely matches the results observed in untreated cells.

In conclusion, under conditions where proliferation is significantly reduced or completely inhibited (Fig. 8
And 9, see this example), expression of the early T cell activation marker CD69 and the IL-2 receptor CD25 remains essentially unaffected; and CD40 levels are comparable to untreated cells. . It should be noted that CD69 is transiently expressed on lymphocytes upon antigen or mitogen stimulation, and continued expression of CD69 requires de novo transcription. Thus, the high CD69 levels observed in treated non-proliferating cells further supports the notion that treatment does not irreversibly inhibit gene expression.

Cytotoxic T Cell Function The effect of S-59 + UVA treatment of T cell lytic function is to generate cytotoxic T cells against gamma irradiation inactivated allogeneic “stimulatory” cells in the MLR. It was evaluated by These cytotoxic T cells were then tested for their ability to lyse < ⁵¹ > Cr labeled target cells obtained from the same donor used to provide inactivated stimulator cells.

(Generation of Cytotoxic T Cells) Peripheral blood (100 ml) was taken from two donors. One is called the "stimulator" and the other is called the "responder". PBMCs were isolated from both populations by Ficoll gradient centrifugation. Responsive PBMCs with a cell density of 5 × 10 ⁶ cells /
Monocytes were depleted by placing them in a tissue culture flask for 1 hour. Stimulated PBMC were gamma-irradiated (2500 cGy) to block cell division. Monocyte depleted responders and irradiation stimulators were separately resuspended in RPMI complete medium at a concentration of 1 × 10 ⁶ cells / ml. An equal volume of responsive cell suspension and stimulator cell suspension is then added to M
Aligned for LR and incubated at 37 ° C. in a 5% CO ₂ incubator for 7 days.

Target Cell Preparation Peripheral blood (50 ml) was collected from the same donor who provided the stimulator cells for the MLR. PBMCs were isolated as above and resuspended in RPMI medium at a concentration of 1 × 10 ⁶ cells / ml, then 2 μg / ml PHA-M (phytohemagglutinin-M) and 10 units / ml recombinant IL. It was stimulated with -2 at 37 ° C for 7 days.

Labeling of target cells with ⁵¹ Cr Target cells were incubated for 2 hours at 37 ° C. in the presence of 200 μCi Na ₂ ⁵¹ CrO ₄ and then washed 3 times to remove unincorporated label. .

Photochemical Treatment of Responsive Cells (PCT) Responder cells (monocyte-depleted as described above) were divided into 4 equal parts, each of which was resuspended in 30 ml PBS containing 1% bovine serum albumin. Suspended. S-59
Three of these portions were added to a final concentration of 0.1, 0.01, or 0.001 μM; the remaining portion served as an untreated control. S-59
Of the sample containing sucrose was transferred to a 30 ml 2410 blood bag, and each of them was mixed with Baxter.
/ Fenwall UVA irradiator at 3 J / cm ² UVA.

CTL Assay The cytotoxic T lymphocyte (CTL) activity of treatment-responsive cell populations was assayed as follows. Treated cells (or control untreated cells) were resuspended in a final volume of 1 ml to a final concentration of 5 × 10 ⁶ cells / ml and 2.5 × 1 of 2-fold serial dilutions.
Made to obtain final concentrations of 0 ⁶ , 1.25 × 10 ⁶ and 0.625 × 10 ⁶ cells / ml. 100 of suspended treatment-responsive cells and each treatment-responsive cell dilution
A μl sample (“effector cells”) was added to each of three wells of a 96 well round bottom microtiter plate. A 100 μl sample of labeled target cells (prepared as described above) (containing 5 × 10 ³ cells) was then added to each well of effector cells, 100: 1, 50: 1, 25: 1 and 12. .5
An effector cell: target cell ratio of 1: 1 was obtained and the mixture was incubated at 37 ° C. for 4 hours. After incubation, the plates were centrifuged at 250 xg for 5 minutes and 100 μl of supernatant was removed from each well. The amount of ⁵¹ Cr in the supernatant was quantified with a γ counter. Average ⁵¹ Cr c for each triplicate set of wells
The pm and standard deviation were calculated.

(Control) Unlabeled target cells were diluted and plated with labeled target cells in the same ratio as effector cells to measure spontaneous release of ⁵¹ Cr. Labeled target cells were also incubated with RPMI and 1% Triton X-100 (3 wells each) and the maximal release of ⁵¹ Cr was measured. Average ⁵¹ Cr cp for each triplicate set of wells
m and standard deviation were calculated.

Results FIG. 13 shows data for release of ⁵¹ Cr at different effector: target (or unlabeled target: labeled target) ratios for treated and untreated leukocytes. These results indicate that cytolytic function is retained by leukocytes treated with doses of S-59 and UVA that block proliferation. Thus, not only non-proliferated treated leukocytes have the same phenotype as untreated cells (as indicated by surface markers and cytokine expression), but they may also have similar functional properties.

In summary, proliferation of treated leukocyte populations, surface marker expression, cytokine synthesis,
And analysis of CTL activity revealed the following characteristics of treated leukocytes: A dose-dependent inhibition of proliferative capacity is observed upon treatment.

2. Cytokine synthesis, cell surface antigen expression, and in vitro cytotoxicity are retained by treated cells.

Accordingly, leukocyte populations, and equivalent cell populations, treated as described herein may facilitate transplantation of donor cells (eg, hematopoietic cells and hematopoietic stem cells) in non-myeloablative hosts. One mechanism by which this can be achieved is by inducing donor-specific tolerance by the treated leukocyte population.

Example 15: Providing GVL effects in the absence of GVHD by treated leukocytes in a mouse model system GHC of donor lymphocytes in an MHC-incompatible B6 / AKR mouse model system.
Experiments were conducted to study the effect of S-59 + UVA treatment on activity. The donor splenocytes were passed through a fine mesh screen to obtain a single cell suspension of unseparated splenocytes, followed by 0.01 μM S-59 and varying doses of UVA (0.5 min, 1 min, 2 min, and 8 minutes). 1 × 10 ⁷ treated donor splenocytes were treated with 5 × 10 ⁶ T
11 with cell depleted bone marrow cells (C57BL / 6, H-2b, Thy1.2 ⁺ ).
AKR, H-2k, Thy1.1 ⁺ hosts subjected to total body irradiation of 00R were injected. Each group of mice (6 per group) was challenged with 250 AKR-M2 leukemia cells 3 days after transplantation. Transplanted animals were observed for clinical signs of GVHD, leukemia recurrence, and death. In addition, the weight of the transplanted animals was recorded every 3-4 days after transplantation.

[0343]   The results summarized in Table 3 and Figures 14 and 15 show that G
For UVA + S-59 effective in reducing VHD while retaining GVL activity
It shows that a suitable range of amounts can be obtained. For example, untreated or lightly treated
(0.5 min UVA + 0.01 μM S-59; irradiation dose = 1.8 J-nM / cm ² ) Addition of lymphocytes to T-cell depleted bone marrow from mild to transplant recipients
Produced severe GVHD (indicated by weight loss in these groups of animals;
(Fig. 14). In contrast, 1 minute UVA + 0.01 μM S-59 (irradiation dose = 3.
7 J-nM / cm²) Were treated with GVL without signs of GVHD.
Was effective in retaining activity (leukemia challenge 70 days later
3 out of 3 survived). See Table 3 and Figure 15. 1 minute UVA +
The body weight of animals treated with 0.01 μM S-59 (FIG. 14) also showed no GVHD.
I showed you that. GVL activity of donor lymphocytes was determined by the same cell S-59 cell concentration.
And higher UVA doses (2 and 8 min UVA, 7.5 and 3 respectively)
0J-nM / cm²) Is equivalent to the irradiation dose)).
Under these conditions, 3 out of 3 mice died within 20 days after transplantation
. See FIG.

[0344]

[Table 3]

Notes: -All groups were irradiated on day -1 and received MHC-incompatible bone marrow transplants and were splenocyte treated on day 0 (where applicable). -The leukemia challenge was done on day 3. -MST = median survival time (days) -Range = date of death (post transplant) (X = survival).

From these results, adding appropriate doses of S-59 + UVA-treated leukocytes to T cell-depleted bone marrow showed that GVHD in a major MHC-incompatible mouse model system.
It can be concluded that Moreover, the addition of properly treated leukocytes facilitates donor transplantation and leads to stable chimerism in MHC-incompatible transplant hosts. Finally, the GVL activity of treated leukocytes is retained. Therefore, S-59 + UVA
Treatment of leukocytes with E. coli is a novel method for modulating the immunological activity of donor lymphocytes to assist allogeneic transplantation.

Example 16: Effect of S-303 on human lymphocyte proliferation and T cell function Measurements were performed on treated allogeneic T cells in the MLR assay. Peripheral blood mononuclear cells (PBMC) were isolated from donors, gamma-irradiated with a dose of 2,500 cGy to prevent autostimulation and used as stimulator cells. PBMCs isolated from different donors were used as responder cells. Responder cells at a concentration of 2 × 10 ⁶ cells / ml were treated with 0.1, 0.2, 0.3, 0.4, or 0.5 μM β-alanine,
Treated with N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester (S-303) for about 15 minutes at room temperature and 2 with PBS containing 1% bovine serum albumin. Washed twice. S-303 treated and untreated responders were co-cultured with gamma-irradiated stimulated cells in a 1: 1 ratio.

Lymphocyte proliferation was performed by adding ³ H thymidine to the co-cultures 6 days after the start of co-culture, harvesting cells on day 7 and measuring ³ H cpm incorporation in the cells. It was measured. Cytokine production was analyzed by sandwich ELISA of culture supernatants collected 24 and 48 hours after the start of co-culture.

Analysis of the proliferative potential of S-303 treated cells (FIG. 16) showed that ³ H thymidine incorporation in S-303 treated allogeneic cells was reduced in cells treated with 0.1 μM S-303, and It is shown to be completely blocked in cells treated with S-303 concentrations above 0.2 μM. Therefore, a dose-dependent inhibition of lymphocyte proliferation was observed in S-303 treated leukocytes.

Gamma interferon (IFN-γ) production was evaluated as a measure of cytokine production by S-303 treated leukocytes. FIG. 17 shows that IFN-γ secretion was 0.1 μM.
And unaffected in cells treated with 0.2 μM S-303. For cells treated with 0.1 μM and 0.2 μM S-303, respectively:
Proliferation is significantly reduced and completely inhibited (see Figure 16).
Cells treated with 0.3 μM S-303, where lymphocyte proliferation was completely inhibited (FIG. 16), produced IFN-γ at approximately 28% of control levels (FIG. 17).

In summary, proliferation of allogeneic stimulated human lymphocytes, measured in MLR, was inhibited in a dose-dependent manner until prior to treatment with S-303. Furthermore, conditions under which its proliferation is completely inhibited (eg treatment of responding cells with 0.2 μM S-303).
Below, the synthesis of IFN-γ remained unaffected. Therefore, S-303 treatment conditions may be obtained that produce lymphocytes that are unable to proliferate upon stimulation and retain immunological activity.

While the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, various changes and modifications may be made without departing from the spirit of the invention. It will be apparent to those skilled in the art that this can be done. Therefore, the above description and examples should not be construed as limiting the scope of the invention.

[Brief description of drawings]

FIG. 1 shows various concentrations of S-59 (squares), AMT (triangles), and 8-MOP.
Shows a decrease in proliferating T cells after PCT with (circles) (UVA = 1 J / cm ²
) (See Example 1).

FIG. 2 shows uptake of ³ H thymidine by effector cells treated with various drug dosages of S-59 in the MLR assay (see Example 2).

FIG. 3 shows the level of IL-2 production in MLR by effector cells photochemically treated with various drug dosages of S-59 (see Example 2).

FIG. 4 shows the level of IFN-γ production in MLR by effector cells photochemically treated with S-59 at various drug dosages (see Example 2).

FIG. 5: PC with different concentrations of S-59 and 0.5 J / cm ² UVA.
The level of IL-8 produced after T is shown as a function of time after treatment (see Example 3).

FIG. 6 shows the level of expression of the CD69 marker after PCT with different doses of AMT and 1 J / cm ² UVA as a function of time after treatment (Example 3).
checking).

FIG. 7 shows psoralen-DNA adduct formation after photochemical treatment with various psoralens in PC. S-59 (square); AMT (triangle); 8-MOP (circle);
9 J / cm ² UVA (see Example 3).

FIG. 8 is measured by ³ H thymidine incorporation in the MLR assay,
3 shows the effect of S-59 and UVA treatment on the proliferation of treated cells. NR
Indicates untreated cells; UV indicates cells irradiated with UVA in the absence of S-59. Also shown are the results obtained when cells were irradiated with ³ J / cm ² UVA in the presence of three different concentrations of S-59. See Example 14 for details.
checking ...

FIG. 9 shows the effect of S-59 and UVA treatment on the proliferation of treated cells, measured after activation of cells treated with anti-CD-3 antibody.

FIG. 10A shows IL-2 production by human PBMCs photochemically treated with S-59 and UVA and stimulated in MLR. Sandwich EL measurement
According to ISA. See Example 14 for details.

FIG. 10B shows IFN-γ production by human PBMCs photochemically treated with S-59 and UVA and stimulated in MLR. The measurement is sandwich E
According to LISA.

FIG. 11A shows CD69 expression in treated and control cells measured at different times (hours) after activation with anti-CD3.

FIG. 11B shows CD25 expression in treated and control cells measured at different times (hours) after activation with anti-CD3.

FIG. 12 shows CD40L expression in treated and control untreated cells measured at different times (hours) after activation with anti-CD3.

FIG. 13 shows ⁵¹ Cr from target cells co-incubated with treated leukocytes.
Cytotoxic T of Photochemically Activated Leukocytes Measured by Release
Shows cell activity. E: T indicates the ratio of effector cells to target cells.

FIG. 14 shows the measurement of mean body weight of irradiated mice that underwent MHC-incompatible bone marrow transplant with S-59 and UVA treated splenocytes, and then challenged with white blood cells for 3 days post-transplant. . Splenocytes can be differentiated for different amounts of time, as shown in the figure.
Exposed to UVA in the presence of 0.01 μM S-59.

FIG. 15 shows an analysis of GVL activity in leukemia challenged mice. The mice received bone marrow transplants with or without injection of treated leukocytes. GV
L activity is indicated by% survival without leukocytes (survival ratios given in brackets).

FIG. 16 shows that after co-culturing with γ-activated allogeneic stimulator cells in MLR,
³ shows an analysis of proliferative activity of S-303 treated responder cells as measured by 7 H thymidine incorporation after 7 days.

FIG. 17 shows the level of IFN-γ in MLR supernatant in responder cells treated with different concentrations of S-303 prior to co-culture.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SZ, UG, ZW), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GE, GH, GM, HR , HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, L V, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U S, UZ, VN, YU, ZW (72) Inventor Taribu, Sohel             United States California 94061,               Redwood City, Camberley               Way 404 (72) Inventor Stasinopros, Adonis             United States California 94568,               Dublin, Shady Creek             Code 7685 (72) Inventor Hay, Derek Jay.             United States California 94521,               Concord, Smoked Treeco             4405 (72) Inventor Hurst, John Yi.             United States California 94717,               Berkeley, Southampton Strike             REIT 101 F-term (reference) 4B065 AA94X BD13 BD34 CA44                 4C084 AA11 NA14 ZA512 ZB072                       ZB262

Claims (37)

[Claims] 1. A method for facilitating an allogeneic transplant procedure comprising the steps of: (a) introducing a population of donor cells into a host, wherein the donor and host are allogeneic to each other. System, and (b) introducing an isolated population of leukocytes into the host; wherein (i) the leukocyte population is 5 per 10 ⁸ base pairs of leukocyte genomic DNA. ~ 1
000 adducts have been subjected to treatment with a compound capable of forming a covalent bond with a nucleic acid; (ii) the leukocyte population is immunologically sufficient to facilitate the implantation of the donor cells. Activity is retained; and (iii) introducing the leukocyte population into the host inhibits the development of graft-versus-host disease. 2. The method of claim 1, wherein the second population is used in donor leukocyte infusion (DLI). 3. The donor cell is selected from the group consisting of hematopoietic cells, myeloid cells, leukocytes, bone marrow cells, islet cells, hepatocytes, nerve cells, cardiomyocytes, mesenchymal cells and endothelial cells. The method according to 1. 4. The method of claim 1, wherein the donor cell comprises an organ or a portion of an organ. 5. A method for the destruction of diseased cells, infected cells, or virulence factors in a host, comprising the steps of: introducing into the host an isolated population of leukocytes, Here, the leukocyte population is 5 to 1 per 10 ⁸ base pairs of leukocyte genomic DNA.
000, which has been subjected to treatment with a compound capable of forming a covalent bond with a nucleic acid such that an adduct is formed. 6. The method according to claim 5, wherein the white blood cells comprise one or more cells selected from the group consisting of T cells, NK cells and antigen presenting cells. 7. The method of claim 5, wherein the diseased cells are cancer cells. 8. The introduction of the leukocyte population provides a leukemic graft effect.
The method according to claim 7. 9. The method of claim 5, wherein the infected cell is infected with a virus. 10. The method of claim 5, wherein the virulence factor is selected from the group consisting of viruses, bacteria, fungi and protozoa. 11. A method for inhibiting the development of graft-versus-host disease in an allogeneic transplant recipient comprising the steps of: introducing into the recipient an isolated population of leukocytes. Wherein the leukocyte population has been subjected to treatment with a compound capable of forming a covalent bond with a nucleic acid such that 5-1000 adducts are formed per 10 ⁸ base pairs of leukocyte genomic DNA.
A method comprising the steps of :. 12. The method of claim 11, wherein the leukocyte population retains sufficient immunological activity to facilitate implantation of the transplant. 13. An isolated population of leukocytes; wherein the leukocyte population is covalently linked to nucleic acid such that 5-1000 adducts are formed per 10 ⁸ base pairs of leukocyte genomic DNA. And a leukocyte population that facilitates allogeneic transplantation procedures, inhibition of graft-versus-host disease, and implantation of a second cell population. A leukocyte population that retains sufficient immunoreactivity to perform one or more functions selected from the group consisting of promoting a leukemic graft effect and destroying diseased cells, infected cells or virulence factors. . 14. The leukocyte population of claim 13, wherein proliferation of at least 90% of T cells in said population is inhibited. 15. The treated leukocytes are IL-1, IL-2, IL-4.
14. The leukocyte population of claim 13, which produces at least 1, 2, 3, 4 and all cytokines selected from the group consisting of IFN-γ, IL-10 and GM-CSF. 16. The treated leukocytes are CD2, CD28, CTLA4.
, CD40 ligand (gp39), CD18, CD25, CD69 (a lymphocyte activation marker) and CD16 / CD56, MHC class I and class II,
CD8, CD4, CD3 / TcR (T cell receptor), CD54 (ICAM-
The leukocyte population according to claim 13, which expresses one or more surface markers selected from the group consisting of 1), LFA-1 and VLA-4. 17. The leukocyte population of claim 13, wherein the leukocytes are stimulated by exposure to diseased cells, antigen presenting cells, antigens or mitogens. 18. A method for preparing an isolated population of white blood cells, comprising:
The following steps: The leukocyte population is expressed as 5 to 1000 per 10 ⁸ base pairs of leukocyte genomic DNA.
Subjecting to treatment with a compound capable of forming a covalent bond with a nucleic acid such that an adduct is formed, such that the leukocyte population facilitates an allograft procedure, One or more selected from the group consisting of inhibition of graft disease, facilitating implantation of a second cell population, promotion of leukemic graft effect and destruction of diseased cells, infected cells or virulence factors. Retaining sufficient immune activity to perform the function. 19. The method of claim 18, wherein the compound comprises a nucleic acid binding moiety capable of non-covalently binding to a nucleic acid. 20. The compound comprises: (a) a nucleic acid-binding moiety; (b) an effector moiety capable of forming a covalent bond with a nucleic acid; and (c) a breakage that covalently bonds the nucleic acid-binding moiety and the effector moiety. 20. The method of claim 19, comprising a facile linker. 21. The nucleic acid binding moiety is an aromatic intercalator,
21. The method of claim 20, wherein the effector portion is mustard. 22. The compound is β-alanine, N- (acridin-9-yl), 2- [bis (2-chloroethyl) amino] ethyl ester (S-303) having the formula: embedded image ] And a salt thereof. 23. The compound contains a photoactivatable moiety that forms a covalent bond with a nucleic acid in response to an electromagnetic stimulus; and the treatment combines the cell with the compound to form a reaction mixture. Exposing the reaction mixture to light to photoactivate the photoactivatable moiety, resulting in the formation of a covalent bond between the photoactive moiety and leukocyte genomic DNA.
19. The method of claim 18, comprising: 24. The compound is furocumarine, actinomycin, anthracyclinone, anthramycin, benzodipyrone, fluorene,
Fluorenones, monostral lipids blue (monostral fats bl)
ue), norphyrin A, organic dyes, phenanthridines, phenazathionium salts, phenazines, phenothiazines, phenylazides, quinolines, thiaxanthenones, acridines and ellipticenes. ,
The method of claim 23. 25. The method of claim 24, wherein the furocoumarin is psoralen. 26. The psoralen is 8-methoxypsoralen (8-MOP), 4
26. The method of claim 25, selected from the group consisting of'-aminomethyl 4,5 ', 8-trimethylpsoralen (AMT), 5-methoxypsoralen, and trioxalen 4,5', 8-trimethylpsoralen. 27. The method of claim 25, wherein the reaction mixture is exposed to light having a wavelength in the range of 200-450 nm. 28. The method of claim 27, wherein the reaction mixture is exposed to light having a wavelength in the range of 320-400 nm. 29. The method of claim 27, wherein the reaction mixture is exposed to ultraviolet light at a dose of 10 ^{−3 to} 100 J / cm ² . 30. The method of claim 29, wherein the reaction mixture is exposed to ultraviolet light for 1 second to 60 minutes. 31. The method of claim 29, wherein the psoralen is present at a concentration in the range of 10 ^{−4 to} 150 μM. 32. The psoralen is 4 '(4-amino-2 having the formula:
-Oxa) butyl-4,5 ', 8-trimethylpsoralen (S-59): 32. The method according to claim 31, which is and a salt thereof. 33. The method of claim 32, wherein the reaction mixture is exposed to ultraviolet light at a dose of ³ J / cm ² . 34. The psoralen is present at a concentration of 15 nM.
The method described in. 35. The method of claim 32, wherein the reaction mixture comprises human serum albumin.
The method described in. 36. The method of claim 35, wherein the human serum albumin is present at a final concentration of 1%. 37. The reaction mixture is 133 μg / ml sodium caprylate, 197 μg / ml sodium acetyltryptophanate and 0.9%.
37. The method of claim 36, which also contains NaCl.