US 6,136,306 AGrant

Method for Treating Tumors

Issue Date:2000-10-24

•21 Claims

•2 Drawing Sheets

Abstract

A method is provided for treatment of a mammalian patient having a tumor by administering to the patient allogenic donor lymphocytes that have been co-cultured in the presence of the patient-derived lymphocytes under conditions sufficient to alloactivate the donor lymphocytes. It is preferred that the donor lymphocytes be introduced intralesionally. This method is preferred for treatment of glioblastoma in humans.

Metadata

Assignee

The Regents of the University of California

Inventor

Gale A. Granger

Application Information

Application Number:US 9439536

Filing Date:1997-10-03

Priority Date:1995-03-17

Art Unit:162

Classifications

IPC:

A61K 3800C12N 508

Field of Search:

435424320.1;240.2;240.2193.21;277.1;9.1;93.3

Patent Drawings (2 sheets)

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for inhibiting tumor cell proliferation. More particularly this invention relates to a method for inhibiting tumor cell proliferation by enhancing the patient's ability to respond immunologically to the tumor.

2. Description of Related Art

Traditional therapies have done little to alter the outcome for patients with high-grade brain tumors, such as glioblastomas, and many other types of tumors, such as systemic melanoma, and cancers of the head and neck. Patients with resectable primary tumors generally experience recurrence of the tumor within one year after surgery, chemotherapy, or radiation. Often these tumors progress rapidly, with or without further conventional therapy. Thus, there is the need to develop new modes of therapy for these deadly tumors.

A new family of cancer therapies developed. in recent years are based on immunotherapy. In general, tumor immunotherapies take one of two approaches: 1) various techniques are employed to activate the patient's immune system to attack the tumor; or 2) the lymphoid cells of the patient are removed and activated by in vitro techniques to produce anti-cancer activity, and the activated cells are then systemically reintroduced into the patient. The clinical effectiveness of these various types of immunotherapy are being evaluated in patients with different types of cancers. However, one of the major problems associated with both of these types of immunotherapy is toxicity observed when immunotherapeutic agents are administered systemically. A method developed to avoid this toxicity is intralesional administration of immunotherapy, for instance by injection directly into the tumor. Intralesional administration of various forms of immunotherapy to cancer patients does not cause the toxicity seen with systemic administration of immunologic agents (M. Fletcher, et al., Lymphokine Res. 6:45, 1987; H. Rabinowich, et al., Cancer Res. 47:173, 1987; S. A. Rosenberg, et al., Science 233:1318, 1989; and G. Pizz, et al., Int. J. Cancer 34:359, 1984).

Recent studies indicated that immunization of animals with tumor cells that were genetically engineered to secrete different cytokines enhanced the induction of a therapeutic immune response. The cytokines are believed to induce a complex set of reactions including: a) increased expression of tumor antigens; b) inflammation and infiltration of the tumor with host lymphoid cells; c) induction of tumor specific immunity; and d) activation of both nonspecific and specific host anti-tumor effector mechanisms, which destroy the tumor. However, although this technique may ultimately prove useful, because it is extremely costly and time consuming, its application may be limited.

Studies in experimental animals (primarily mice) show that the chronic release of cytokines within a tumor may induce a host anti-tumor response and tumor regression. Repeated intralesional injection of cytokines such as Interleukin-2 (IL-2), Tumor Necrosis Factor (TNF) and Interferon-.gamma. (INF-.gamma.) has been shown to cause regression of cutaneous sarcomas (S. P. Creekmore, et al., Resident and Staff Physician 34:23-31, 1988; P. Greenberg, et al., Basic and Tumor Immunology (R. Herberman, ed.) p. 302, 1983; E. Grimm et al., Lymphokines, 9:279-311, 1984; G. Forni, et al., Lymphokines 14:335-360, 1987). It has also been shown that injection into a tumor of the animal's tumor cells that have been genetically engineered to secrete cytokines such as IL-2, IL-4, TNF and Granulocyte Monocyte Colony Stimulating Factor (GM-CSF) will induce host anti-tumor immunity (E. Feron, et al., Cell 60, 397-403, 1990; P. Galumbek, et al., Science 254:713-716, 1991; A. Ascher, et al., J. of Immunol. 146:3227-3234, 1991). These latter results have been obtained even in treatment of tumors that were previously thought to be non-immunogenic.

J. M. Redd, et al. (Cancer Immunology and Immunotherapy 34(5):349, 1992) have shown in rats that allogeneic lymphocytes sensitized against donor alloantigens can inhibit tumor formation when co-injected into the brain of a rat with 9L glioblastoma. In a separate study, both normal and alloimmune spleen cells from Wistar rats were injected into established 6-day T9 brain tumors in the Fischer rat. Intralesional injection of normal Wistar spleen cells from Wistar rats, previously immunized against Fischer alloantigens, cured the tumors in 50% of the Fischer rats. In contrast, untreated animals and non-responders died within 30 days. Survivors appeared completely normal and intracranial injections of allogeneic cells into normal rats caused no detectable change in behavior or survival over a three month period. Histopathologic examination of brains from treated tumor bearing animals revealed: a) mononuclear cell infiltration, massive tumor necrosis beginning at 2 to 4 days and total tumor destruction by 15 days; or 2) cellular infiltration, early tumor destruction and then tumor regrowth progressing to death of the animal. No damage to normal brain tissue was evident at any time in these animals. Tumor regressor animals developed systemic immunity, for they proved totally resistant to intracranial rechallenge with viable tumor. Although these results in rats are of interest, their value in reasonably predicting what would be seen in a highly unrelated species, such as a human, is highly questionable in view of the considerable species diversity which exists, especially with respect to the immunological response to tumors.

Human glioma patients with localized and surgically accessible tumors are logical candidates for intralesional immunotherapy. Multiple Phase I studies in adult patients with gliomas have been reported employing intratumor implants of autologous peripheral blood lymphocytes activated in vitro with IL-2. While little clinical effect was noted, negative side effects were few and occurred only when excessive levels of IL-2 were co-administered with the cells (K. S. Jacobs, et al., Cancer Res., 47:2101, 1986; R. Merchant, et al., Neurosurgery 23:725, 1988). Studies of these patients revealed that survival correlated directly with the ability of implanted cells to secrete the cytokine TNF. The discovery of inhibitors for both TNF and IL-1 in the serum or tumor cyst fluid, and in primary cultures of the tumors from these patients suggests the tumor cells surround themselves with agents that block the host anti-tumor response. These inhibitors may prevent cytokine activated implanted cells from remaining active in the tumor long enough to cause its destruction. This concept is supported by findings in brain tumor studies in rats. When IL-2 active lymphoid cells were implanted into C6 and T9 glioma brain tumors in Wistar and Fischer rats, respectively, histopathologic examination revealed the implanted IL-2 activated lymphoid cells only remained in the tumor site for 4 to 6 days (W. Carson et al., J. of Immunotherapy 10(2):131-140, 1991).

The mechanisms operative in causing tumor regression in the animals treated with allogeneic lymphoid cells include a graft vs. host and host vs. graft reaction in the tumor site. These powerful immunologic reactions may stimulate high levels of endogenous cytokine production in the tumor, overcome local levels of cytokine inhibitors, and presumably stimulate infiltration, recruitment and activation of both specific and non-specific host anti-tumor activity. The tumor regressor animals were found to be resistant to tumor rechallenge. However, it has heretofore been unknown whether any treatments based on similar methods would achieve similar results in human subjects sufficient to be considered effective in the treatment of human tumors.

Therefore, in view of the limitations of the prior art, new and better methods for treating mammalian tumors are needed. In particular, new methods of intratumor immunotherapy are needed for human cancer patients for whom regression of an individual solid tumor could prove life saving.

SUMMARY OF THE INVENTION

The present invention provides a method for inhibiting tumors in mammals. In the general practice of this invention, the tumor patient's peripheral blood mononuclear cells (PBMCs) are co-cultured in vitro to induce a mixed lymphocyte cell reaction with healthy lymphocytes derived from a normal donor, preferably an allogeneic donor. Preferably the normal lymphocytes are from a donor unrelated to the tumor patient, and preferably the normal lymphocytes are obtained by leukapheresis from whole blood. During co-culture the allogenic donor lymphocytes are specifically activated against the patient's alloantigens. The mixture of live alloactivated donor and patient lymphocytes produced by mixed lymphocyte culture are referred to herein as "MLCs." The MLC activated cells produce a mixture of cytokines which have been shown to induce a primary immune response in vitro. In the treatment of glioblastoma, for instance, the patient is a human and the alloactivated lymphocytes are surgically implanted into the patient's brain at the primary tumor site, optionally together with the patient's PBMCs from the co-culture as a MLC mixture, to induce the patient's immune system to attack autologous tumor cells. In the treatment of other types of tumors, the MLCs are injected into a tumor site, site of metastases, or body cavity, such as the peritoneum. Optionally, the MLCs are administered peripherally, such as at a non-primary tumor site, in the treatment of tumors other than glioblastoma.

In one embodiment of the invention, the alloactivated donor lymphocytes obtained by co-cultivation with patient-derived lymphocytes are isolated from the MLC mixture and administered intralesionally as an implant placed directly into the tumor of a patient desiring protection against recurrence of a tumor, for instance in the proximity of a surgically debulked tumor, or desiring treatment of an inoperable tumor. Alternatively, the alloactivated donor lymphocytes can be administered peripherally in treatment of a tumor, such as at a secondary or metastatic tumor site. In another embodiment, the allogenic activated lymphocytes are co-cultivated with patient-derived lymphocytes, and the mixture of MLCs is implanted or introduced peripherally as a mixture.

A BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows survival times of glioblastoma patients treated with intratumor implant of MLC cells.

FIG. 2 is a graph showing MRI scans indicating the reduction in tumor size in nine glioblastoma patients, two patients, one receiving a single implant dose of 4.times.10.sup.9 MLCs and one receiving a single implant dose of 6.times.10.sup.9 MLCs, have continued to date to show a progressive reduction in tumor mass over periods of 58 and 74 weeks, respectively.

A DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating tumors in mammals using an allograft of donor lymphocytes that have been co-cultured in vitro in a mixed lymphocyte (MLC) reaction with the patient's mononuclear cells, preferably from peripheral blood, to activate the donor lymphocytes against antigens associated with the patient's tumor. It is believed that a graft v. host response and a host v. graft response to the allograft induce a powerful immunologic reaction in the tumor site that results in cytokine production and tissue destruction. During this reaction the host lymphoid cells identify both the graft lymphoid cells and tissue as foreign and both are inhibited or destroyed by local and systemic immune response. This host reaction occurs not only at the implant site of the graft lymphoid cells, but may also occur peripherally, such as at a secondary or metastatic tumor site. In addition, the implanted, donor lymphocytes immunologically attack the tumor in a graft v. host response. Thus, a strong immunologic reaction in the center of the tumor produces an environment that overcomes the cytokine inhibitors that may be present at the tumor site. The increase in immunologic reaction will inhibit growth, decrease size and/or eradicate established tumors or developing metastases, as well as inhibit recurrence at the site of surgically debulked tumors. Thus, the present invention provides a method for potentiating the systemic immune response to the patient's tumor by introducing into the patient a viable preparation of the alloactivated cells described herein.

Numerous studies have shown that the in vitro environment in a mixed lymphocyte reaction facilitates an active primary immune response to allogeneic and tumor associated antigens (M. Gately, et al, JNCI 69:1245, 1982; S. Lee, et al., J. Experimental Medicine 147:912, 1978; J. Zarling, et al., Nature 262:691, 1976). In the present invention, the MLC reaction occurs within the patient's own tumor tissue, thereby stimulating the patient to respond against its own tumor. Preferably the co-culture is performed, generally from 1 to 5 days. Since release of key cytokines takes place during the early stages of the co-culture, it is preferred that the co-cultured cells be implanted in the early stages of the co-culture reaction, usually during the first 48 hours of the reaction when cytokine levels are normally highest. This method results in release of key cytokines directly into the tumor tissue so that an environment is created that is conducive to antigen recognition and development of cell mediated immunity directed against the antigen. Standard techniques, such as those based on immunoassay, can be used to measure the level of various cytokines, including TNF, LT and gamma interferon, present in the MLC culture supernatants. Levels of TNF and LT vary from 50 to 150 units of biologic activity/ml or 500 to 3500 pg/ml of supernatant. As a result of the in vitro co-culture, healthy allogeneic lymphoid cells from a donor are specifically activated against the patient's alloantigens.

The implanted cells are generally allowed to come into direct contact with tumor cells. Thus, as used herein the term "implant" and "implanted" means that the cells are placed into the patient's body as a group, optionally within a thickening matrix of coagulated serum or other suspension or gel-like substance.

Examples of tumors that can be treated by the method of this invention include the following:

Brain tumors, such as astrocytoma, oligodendroglioma, ependymoma, medulloblastomas, and PNET (Primitive Neural Ectodermal Tumor);

Pancreatic tumors, such as pancreatic ductal adenocarcinomas.

Lung tumors, such as small and large cell adenocarcinomas, squamous cell carcinoma, and bronchoalveolarcarcinoma;

Colon tumors, such as epithelial adenocarcinoma, and liver metastases of these tumors;

Liver tumors, such as hepatoma, and cholangiocarcinoma;

Breast tumors, such as ductal and lobular adenocarcinoma;

Gynecologic tumors, such as squamous and adenocarcinoma of the uterine cervix, and uterine and ovarian epithelial adenocarcinoma;

Prostate tumors, such as prostatic adenocarcinoma;

Bladder tumors, such as transitional, squamous cell carcinoma;

Tumors of the RES System, such as B and T cell lymphoma (nodular and diffuse), plasmacytoma and acute and chronic leukemia;

Skin tumors, such as malignant melanoma; and

Soft tissue tumors, such as soft tissue sarcoma and leiomyosarcoma.

In one embodiment of the method of this invention, the alloactivated donor lymphocytes are implanted at the tumor site, optionally together with the patient's PBMCs from the co-culture, to supplement and enhance the patient's immune system attack upon autologous tumor cells. For instance, in one embodiment of the invention alloactivated human donor lymphocytes obtained by co-cultivation with patient-derived PBMCs or cell lysates of PBMCs are administered intralesionally in a human, for instance as an implant placed directly into the brain of a patient desiring protection against recurrence of a glioblastoma tumor. The alloactivated donor lymphocytes can be implanted in the proximity of a surgically debulked tumor, or a tumor treated by irradiation, chemotherapy, or other appropriate techniques. In another preferred embodiment, the allogeneic activated lymphocytes are co-cultivated with patient-derived lymphocytes and the mixture of co-cultured allogenic and autologous cells, known herein as "the MLCs," is implanted intralesionally.

The typical implant comprises a therapeutic amount of the allostimulated donor lymphocytes. As used herein the term "a therapeutic amount" means a sufficient quantity of MLCs or donor allostimulated lymphoid cells obtained from the mixed lymphocyte culture to inhibit growth, decrease the size of and/or eradicate established tumors, and/or prevent tumor recurrence at the site of a tumor that has been surgically debulked or treated with chemotherapy or irradiation. More generally, a therapeutic amount may vary with the potency of each batch of alloactivated donor cells; the amount required for the desired therapeutic or other effect, the mode of administration, i.e., whether by direct implant into a tumor or body cavity or by peripheral administration, such as intravenously; and the rate of elimination or breakdown of the MLCs by the body once implanted or administered. In accordance with conventional prudent formulating practices, a dosage near the lower end of the useful range may be employed initially and the dosage increased or decreased as indicated from the observed response, as in the routine procedure of the physician. In general, however, a unit dosage for direct implant comprises from about 2.times.10.sup.9 to about 6.times.10.sup.9 MLCs. For instance, it has been discovered that in the treatment of brain tumors, the upper limit of cells that can be implanted is about 6.times.10.sup.9. Alternatively, a unit dosage for peripheral administration usually comprises from about 2.times.10.sup.9 to about 2.times.10.sup.10 MLCs.

The invention further comprises a sterile vial or other container holding a composition comprising a unit dosage of the MLCs. Typically, the vial or container will bear a label sets forth information concerning the pharmaceutical use of the composition in treating a tumor in a human, such as FDA approval for use of the composition in the treatment of a human having one or more of the tumors against which the method of treatment of the invention is effective as described herein.

Although any known method of obtaining PBMCs from a donor can be used, it is preferred to obtain approximately 150 to 300 ml of leukapheresis suspension containing the donor PBMCs, utilizing techniques of leukapheresis that are well known in the art for supportive apheresis according to the instructions of the manufacturer of the leukapheresis equipment. For instance, leukapheresis can be performed using a Cobe 2997, Cobe Spectra.RTM. (Lakewood, Colo.), Fenwall CS 3000 (Deerfield, Ill.), or Haemonetics (Braintree, Mass.) blood cell separator. Generally, a flow rate of 40 to 50 ml/min for 2 to 4 hours with lymphocyte yield of 2-4.times.10.sup.9 can be used to process a total donor blood volume of 7,000 to 12,000 ml to yield 200 to 250 ml of leukapheresis suspension having less than 1 ml of red cells. For example, if a Cobe 2997 blood cell separator is used, the centrifuge rate is generally about 5.times.g, the flow rate is up to about 45 ml/min and the collection rate is no more than or equal to 2.5 ml/min. One skilled in the art will appreciate that the yield of lymphocytes will vary with the donor and the leukapheresis machine used. For instance, if the donor pre-absolute lymphocyte counts are in the 0.6.times.10.sup.9 to 1.0.times.10.sup.9 level, as little as 150 ml of donor leukapheresis suspension can be drawn.

If desired, the donor cells can be contacted with a stimulatory cytokine such as IL-2 to trigger activation of the cells to the patient-derived antigen during co-culture and to further stimulate lymphocyte proliferation.

Donor PBMCs are obtained from the donor blood fraction, exercising care to avoid rupture of mononuclear cells, for instance by centrifuging the blood fraction containing the mononuclear cells through a cell separation medium such as Histopaque.RTM. 1.077 at 350.times.g for 7 to 10 minutes. Those of skill in the art will know of other PBMC cell separation techniques that can readily be utilized. Donor blood is typically pre-screened 3-7 days prior to surgery for HIV, Hepatitis A, B, and C, and VDRL.

Sufficient anticoagulant, such as 2% citrate, is added to the donor and patient blood or blood fraction to prevent coagulation upon withdrawal. Alternative anticoagulants and mixtures thereof known to one of skill in the art can also be used, such as anti-coagulant citrate dextrose formula A (ACDA) 15 ml/citrate 100 ml; anti-coagulant citrate dextrose formula B (ACDB) 25 ml/citrate 100 ml; or citrate phosphate dextrose (CPD) 14 ml/citrate 100 ml.

Typically, whole blood is withdrawn from the patient to be treated according to the invention using methods known in the art, such as vena puncture. PBMCs are isolated from patient whole blood, usually by centrifugation through a cell separation medium, such as Histopaque.RTM. 1.077 (Sigma, St. Louis, Mo.), and are thoroughly washed to free the cells of the clotting factor in the patient's blood.

Samples of both the donor's and patient's blood or blood fraction should be thoroughly tested to ensure sterility before co-culturing of the cells. Typical of the tests for sterility of blood components that can be conducted by one of skill in the art are those using such growth media as thioglycollate broth, tryptic soy broth and Roswell Park Memorial Institute Tissue Culture Medium (RPMI) with 10% heat-inactivated fetal bovine serum (FBS)(RPMI--10%) and 1% L-glutamine with no added antibiotics. Sterility tests utilizing the culture of blood cells in such growth media are illustrated in Example 1 of this application. Alternative sterility tests will be known to those of skill in the art.

Before mixed lymphocyte culture can be performed, typically the PBMCs isolated from whole blood are analyzed to determine the number of live cells per unit volume. This can be performed, for example, by using a stain that differentiates between living and dead cells and counting the cells in a Neubauer chamber. Typical stains for this use are trypan blue and Eosin Y dyes, both of which can be used in wet preparations. Alternative stains will be known to those of skill in the art. Generally, the concentration of live cells is standardized by diluting the preparation of PBMCs to achieve a predetermined concentration of live cells per unit volume. Although one skilled in the art may select a number somewhat higher or lower, it is generally preferred that the number of live cells be fixed at a concentration of about 10.sup.7 cells/ml for the purposes of conducting the mixed lymphocyte culture. The donor cells are typically cultured at a ratio of 10:1 to 20:1 compared to patient cells.

Standard techniques for conducting mixed lymphocyte culture using mammalian (i.e., human) cells are well known in the art and are illustrated in Example 1 of this application. See, for instance, Current Protocols in Immunology, Ed. J. E. Coligan, et al., John Wiley & Sons, Inc., 1994, Sec. 7.10 and M. Gately, et al., supra; S. Lee, et al., supra; and J. Zarling, et al., supra, which are incorporated herein by reference in their entirety. To block response of the patient stimulator cells to the donor responder cells (back stimulation), it is preferred that the patient cells be irradiated or treated with a DNA binding agent, such as mitomycin C, before mixture of the cells during co-culture to reduce or eliminate patient cell proliferative potential, as is well known in the art. In the present invention, donor lymphocytic cells are typically co-cultured in a short term mixed lymphocyte culture with the patient's PBMCs for a period of at least 48 hours, and preferably from 1 to 5 days. As a result of the in vitro co-culture, healthy lymphoid cells from the unrelated donor are specifically activated against the patient's tumor associated antigens.

Preparations for parenteral or intravenous administration are typically contained in a "physiologically compatible carrier." Since the cells utilized in the practice of this invention are live, a physiologically compatible carrier is one that does not impair viability of the cells, i.e., is hypotonic and at physiological pH. Such carriers include sterile aqueous salt solutions, suspensions and emulsions, including saline and buffered media, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's solution. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. For administration by non-intravenous routes, the carrier can be in the form of clotted plasma, preferably the patient's clotted plasma. Alternatively the carrier can be a plasma-free, physiologically compatible, biodegradable solid or semi-solid, such as a gel, suspension or water soluble jelly. Acacia, methylcellulose and other cellulose derivatives, sodium alginate and tragacanth suspensions or gels are suitable for use as carriers in the practice of this invention, for example, sodium carboxymethylcellulose 2.5%, tragacanth 1.25% and guar gum 0.5%.

In a preferred method of implant at a tumor site, the co-cultured lymphocytes are collected from the co-culture supernatant (after at least 48 hours of co-culture) by centrifugation at the time of surgery. The collected cells are washed two times with injectable saline and re-suspended in platelet free, decalcified plasma obtained from the patient the previous day. The cells in plasma are transported to surgery, the plasma is re-calcified by the addition of calcium, preferably in the form of calcium gluconate so that the plasma clots, embedding the cells in an autologous plasma clot. The patient's tumor is then surgically debulked, and the clot is aseptically minced and implanted into the tumor site.

The following examples illustrate the manner in which the invention can be practiced. It is understood, however, that the examples are for the purpose of illustration and the invention is not to be regarded as limited to any of the specific materials or conditions therein.

EXAMPLE 1

A. Mixed Lymphocyte Cell (MLC) Culture Procedure

1. Collection of Responder PBMC from Unrelated Donor

Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis from normal healthy donors unrelated to the patient. Donors were pre-screened to test for complete blood count (CBC) with differential, Hepatitis A, B, and C, VDRL, and HIV-I.

Approximately 150 to 300 ml of leukapheresis suspension containing PBMC was collected from each donor, using standard blood donation procedures for supportive apheresis according to the manufacturers' instructions. The leukapheresis was performed using a Fenwall CS 3000 (Deerfield, Ill.) blood cell separator. A flow rate of 40 to 50 ml/min for 2 to 4 hours with lymphocyte yield of 2-4.times.10.sup.9 processed a total donor blood volume of 7,000 to 12,000 ml to yield 200 to 250 ml of leukapheresis suspension having less than 1 ml of red cells. If a Cobe 2997 blood cell separator was used, the centrifuge rate was 5.times.g, the flow rate was up to 45 ml/min, and the collection rate was no more than or equal to 2.5 ml/min.

However, if donor pre-absolute lymphocyte counts were in the 0.6.times.10.sup.9 to 1.0.times.10.sup.9 range, as little as 150 ml of leukapheresis product was drawn. Hematocrit for the final product was 3.5%. At least one total blood volume was processed for 80% efficiency of lymphocyte collection.

The anticoagulant used was either 2% citrate or a citrate/anticoagulant ratio of ACDA--15 ml/citrate--100 ml; ACDB--25 ml/citrate--100 ml; or CPD--14 ml/citrate--100 ml. To obtain the utmost product purity, the actual and final product from the cell separator was transported as a pure concentrate of cells in autologous plasma. The cells were not washed, and no albumin was added.

2. Preparation of Donor Cells

The leukapheresis product was transported to the MC Oncology Research Laboratory for the production of allogeneic mixed lymphocyte cells (MLCs) for immunotherapy.

Cells were drained from the leukapheresis pack into two or three 250 ml centrifuge tubes, removing and setting aside 3 ml for sterility tests to be done during centrifugation. Cell concentrate was diluted with phosphate buffered saline (PBS) and centrifuged for 7 minutes at 2,000 rpm. Centrifugation was repeated twice for a total of three times to wash the cells free of the clotting factor in the donor's serum.

Three 1 ml aliquots from the 3 ml removed from the leukocyte suspension were placed into sterile capped tubes for sterility testing. The first 1 ml aliquot was added to thioglycollate medium (Difco, Detroit, Mich.) (30-35.degree. C., 48 hr.); a second 1 ml was added to tryptic soy broth (Difco, Detroit, Mich.) (25-30.degree. C., 48 hr.); and the third 1 ml was added to RPMI 1640 (GIBCO, Gaithersburg, Md.) with 10% heat-inactivated FBS (RPMI--10%) and 1% L-glutamine, but without antibiotics.

Cells were spin washed twice at 150 g for 10 minutes in PBS to remove platelets. The supernatant was very carefully discarded as cells were in a slurry and not a pellet. Cells were resuspended in AIM V (GIBCO, Gaithersburg, Md.) supplemented with 2% heat inactivated FBS (2% AIM V) to 420 ml, and placed into a T-175 cm.sup.2 flask.

Patient or donor blood was diluted 1:1 with sterile saline. For cell separation, 35 ml of cell suspension was carefully layered onto 15 ml Histopaque.RTM. 1.077 suspension medium (Sigma, St. Louis, Mo.) in each 50 ml tube and centrifuged at 250 g for 45 minutes. Centrifugation was started slowly and gradually increased to full speed. After centrifugation, the interface containing mononuclear cells between the Histopaque.RTM. suspension medium and the plasma layer was carefully collected with a 25 ml sterile pipet, deposited into clean 50 ml centrifuge tubes, diluted with 2% AIM V Media 1:1, and centrifuged at 550 g for 7 to 10 minutes to form a cell pellet. Cells remained a minimum of time in the Histopaque.RTM. suspension medium, because it is toxic to the cells.

The supernatant was discarded, the pellet was resuspended in 2% AIM V and divided into two 50 ml centrifuge tubes to a total volume 40 ml, and centrifuged at 550 g for 5 minutes. After washing, the supernatant was discarded. The washing step was repeated twice for a total of three times. After the last wash, cells in each tube were resuspended in 50 ml of 2% AIM V. Aliquots of 1 ml of the resuspended cells were diluted to a ratio of 1:10 in 2% AIM V per tube, then further diluted 1:1 in Trypan Blue (Sigma, St. Louis, Mo.) to distinguish dead from live cells, and the live cells were counted in a hemocytometer. Cells were set at 2.times.10.sup.6 /ml with 2% AIM V.

3. Collection of Stimulator PBMC from Tumor Patients.

From 200 to 400 ml of peripheral blood cells were drawn from each glioblastoma patient by vena puncture and placed into 250 ml centrifuge tubes, removing and setting aside 3 ml for sterility tests to be done during spinning. Blood cells in the centrifuge tubes were diluted with saline and centrifuged for 7 minutes at 550 g. Centrifugation was repeated twice for a total of three times to wash the cells free of the clotting factor in the patient's serum. Sterility testing was conducted as described above.

Cells were washed twice by centrifugation at 150 g for 10 minutes in saline to remove platelets, the supernatant was very carefully discarded, and 420 ml of cells were resuspended in a T-175 cm.sup.2 flask in saline.

15 ml of Histopaque.RTM. 1.077 cell separation medium was added to twelve 50 ml centrifuge tubes, and 35 ml of cells suspended in saline were layered onto the Histopaque.RTM. 1.077 in each 50 ml tube. The cell suspensions were spun at 250 g for 45 minutes, starting centrifugation slowly and gradually increasing speed.

After centrifugation, the mononuclear cells at the interface between the Histopaque.RTM. cell separation medium and the plasma layer were carefully collected with a 25 ml sterile pipet into 2 sterile 250 ml centrifuge tubes and diluted with 2% AIM-V to a final volume of 250 ml. The diluted mononuclear cells were centrifuged at 550 g for 7 to 10 minutes. For washing, the supernatant was discarded, then the cell pellet was re-suspended with 2% AIM V and centrifuged at 550 g for 5 minutes. The washing step was repeated for a total of three times.

After the last washing step, cells were re-suspended in 50 ml of 2% AIM V, 1 ml of the cell suspension was diluted 1:10 in 2% AIM-V per tube, and the number of viable cells was determined by enumeration in a 1:1 in Trypan Blue as described above.

The above-described procedures for collection of cells and proof of sterility have been approved by the State of California for GMP for sterility and quality control.

4. Alloactivation of Patient Mononuclear Cells (PBMC) with Donor Leukocytes

The isolated patient PBMCs were re-suspended at 10.sup.7 cells/ml in AIM-V, 50 .mu.g Mitomycin C (Bristol-Mayer Squibb, Princeton, N.J.) were added per ml of patient cell suspension, and the suspension of PBMCs was incubated at 37.degree. C. for one hour to block response of the stimulator cells to the responder cells (back stimulation). After one hour of incubation, the excess mitomycin C was washed from the cells by alternate centrifugation (250 g for 5 min), and the cells were resuspended in AIM-V. After mitomycin treatment of the patient's PBMCs, the cells were added at a 20:1 to 10:1 donor:patient cell ratio to the donor culture (obtained as described above).

For co-culture, the donor and mitomycin C-treated patient PBMC suspension was placed in a sealed sterile Fenwal tissue culture system especially designed for culture of PBMC for reimplantation into patients. Cells were passed in sealed systems via Fenwal cell transfer units and pumps according to the manufacturers instructions, and cultured in a 37.degree. C. incubator for 48 hours.

5. Sterility Testing of Alloactivated Cells

Two days prior to implantation of the cell suspension, the following three sterility tests were performed. 10 ml sterile aliquots were removed from each tissue culture bag, placed into sterile capped 15 ml centrifuge tubes, and centrifuged for 10 minutes at 450 g. In each tube, the pellet was resuspended in 3.0 ml of PBS. A 1 ml aliquot of the cell suspension was added to each of three sterile capped tubes containing 2 ml of thioglycollate broth, tryptic soy broth, or RPMI--10% and incubated for 48 hours. Each cell suspension was examined microscopically prior to implant to detect signs of microbial growth.

On the day of surgery, the cells were centrifuged out of their medium, washed two times with saline and re-suspended in platelet free, decalcified plasma obtained from the patient the previous day. The cells were transported to the operating room in plasma, then the plasma was re-calcified by the addition of calcium gluconate so that it clots just before implantation into the tumor bed.

The day of surgery a drop of collected cell pellet was again examined for sterility under the microscope. Just prior to clotting, a 100 .mu.l aliquot of the cell suspension was added to 2 ml each of RPMI--10% without antibiotics, thioglycollate and tryptic soy broth in a sterile capped tube. The samples were then incubated for four days after surgery, and a running log was kept of this last sterility test.

Cyto-implants produced by the above described method have received FDA approval for use in human cancer patients (IND-BB 6288).

B. Intralesional Implant of Mixed Lymphocyte Cell Culture

A human Phase I trial was initiated to test the effects of intracranial injection of alloactivated allogeneic lymphoid cells in patients with recurrent glioblastoma. A total of 9 patients with recurrent Grade 3 and Grade 4 glioblastoma were entered into this trial. MLCs obtained from mixed lymphocyte culture in Step 3 above were implanted intralesionally in the debulked tumor at the time of surgery. Three patients were tested at each of three cell doses of 2.times.10.sup.9, 4.times.10.sup.9, and 6.times.10.sup.9 of MLCs. The patients were followed clinically and magnetic resonance imaging (MRI) scans were performed at various monthly intervals on each patient following implantation to monitor the progress of the disease.

All patients complained of headache and nausea for 1 to 2 months after surgery. Patients receiving the highest cell dose experienced the highest number of symptoms. Four of the five patients, including all those in the highest dose and one each in the lower doses, showed a 50% or greater reduction in the area of tumor enhancement in MRI scans over a 1 to 3 month period after the implant. The results of MRI scans are shown in FIG. 1, which measures survival time of glioblastoma patients treated with intratumor implants of MLC cells. Two patients, one receiving a single implant dose of 4.times.10.sup.9 MLCs and one receiving a single implant dose of 6.times.10.sup.9 MLCs, have continued to date to show a progressive reduction in tumor mass over periods of 58 and 74 weeks, respectively. These two patients also have shown a marked improvement in Karnofsky score. The results of MRI scans are shown in FIG. 2, which measures the reduction of tumor volume as a function of time since implant of MLCs.

EXAMPLE 2

Phase I studies have been conducted in two patients with systemic melanoma. Mixed cultures of patient and donor lymphocytes were prepared as described in Example 1. The patients were treated by intralesional injection of 2.times.10.sup.9 MLC cells into cutaneous tumors. Neither patient exhibited negative effects. The first patient experienced necrosis and destruction of the injected cutaneous tumor and inflammation at a distant metastasis. The second patient showed inflammation at the injected tumor, but no necrosis at the present dosage level. Treatment of these patients is ongoing.

EXAMPLE 3

A human Phase I clinical study has been conducted to test the effect of intratumor implant of MLC cells in patients with pancreatic cancer. Four patients with incurable, untreatable pancreatic cancer received intralesional implant of 4.times.10.sup.9 MLC cells . Little or no toxicity was found. A reduction in tumor mass occurred and extension of life span has been observed. Three patients have experienced a greater than 50 percent reduction in tumor mass. In patient No. 3, the serum levels of CA-19-9, a marker for this tumor, declined from a high of 206 to 86 over a two month period. Seven months after treatment, two of the treated patients have returned to their jobs, have no clinical symptoms of tumor, and are living a totally normal life.

The foregoing description of the invention is exemplary for purposes of illustration and explanation. It should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following claims are intended to be interpreted to embrace all such modifications.

Claims

What is claimed is:

1. A method of treating a human patient having a tumor, comprising implanting at or around the site of a solid tumor in said patient a cell population comprising alloactivated human donor lymphocytes, wherein said alloactivated human donor lymphocytes are produced by coculturing human donor lymphocytes ex vivo with leukocytes from said human patient, wherein the tumor is selected from the group consisting of melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer, and breast cancer, and whereby said patient is treated.

2. The method of claim 1, wherein the treatment is effective in inhibiting tumor growth.

3. The method of claim 1, wherein the treatment is effective in improving survival time.

4. The method of claim 1, wherein the tumor is a melanoma.

5. The method of claim 1, wherein the tumor is a pancreatic cancer.

6. The method of claim 1, wherein the tumor is a pancreatic ductal adenocarcinoma.

7. The method of claim 1, wherein the tumor is a liver cancer.

8. The method of claim 7, wherein the tumor is a hepatoma.

9. The method of claim 1, wherein the tumor is a colon cancer.

10. The method of claim 7, wherein the tumor is a liver metastasis.

11. The method of claim 1, wherein the tumor is a prostate cancer.

12. A method for preparing a pharmaceutical composition containing alloactivated human donor lymphocytes for the treatment of cancer, comprising the steps of: a) obtaining leukocytes from a human patient having a tumor selected from the group consisting of melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer, and breast cancer; b) coculturing human donor lymphocytes ex vivo with the leukocytes so as to alloactivate the donor lymphocytes; and c) preparing the cocultured cells for human administration at a time when the cocultured cells, upon implantation at or around the site of a solid tumor in the patient, are effective in the treatment of the tumor.

13. A pharmaceutical composition prepared according to the method of claim 12.

14. The composition of claim 13, wherein the pharmaceutical composition comprises about 2.times.10.sup.9 to about 2.times.10.sup.10 of the cocultured cells.

15. A method for preparing a pharmaceutical composition containing alloactivated human donor lymphocytes for the treatment of cancer, comprising the step of coculturing human donor lymphocytes ex vivo with leukocytes from a patient having a tumor so as to alloactivate the donor lymphocytes, and harvesting the cells from culture at about the time when gamma interferon (.gamma.-IFN) secretion by the cultured cells is highest, wherein the pharmaceutical composition, upon implantation at or around the site of a solid tumor in the patient is effective in the treatment of the tumor.

16. The method of claim 15, wherein the harvesting is at a time which is about 48 hours after initiation of the culture.

17. A pharmaceutical composition prepared according to the method of claim 15.

18. A pharmaceutical composition prepared according to the method of claim 16.

19. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition comprises about 2.times.10.sup.9 to about 2.times.10.sup.10 of the cocultured cells.

20. A method of treating a human patient having a tumor, comprising implanting at or around the site of a solid tumor in the patient a pharmaceutical composition according to claim 17.

21. A method for inducing a specific immune response against a tumor in a human patient, comprising implanting at or around the site of a solid tumor in the patient a pharmaceutical composition according to claim 17.

Patent Citations (8)

Patent	Date	Inventor
US4038145	1977-07-01	Devlin
US4677056	1987-06-01	Dupont et al.
US5057423	1991-10-01	Hiserodt et all
US5308626	1994-05-01	Landucci et al.
US5382427	1995-01-01	Plunkett et al.
US5484596	1996-01-01	Hanna, Jr. et al.
CAX1297002	1992-03-01
WOXWO95/31107	1995-11-01

Non-Patent Literature (61)

Declaration by Gale A. Granger regarding Human Clinical Trials.
Appendix B (item 1): E.W.B. Jeffes et al. Therapy of recurrent high grate gliomas with surgery, and autologous mitogen activated IL-2 stimulated killer (MAK) lymphocytes . . . J. Neuro-Oncol. 15:141-155, 1993.
Appendix B (item 3): G. Ioli et al. "Basic & clinical studies with intratumor immunotherapy of gliomas with allogeneic lymphoid cells" Proc. Amer. Assoc. Cancer Res. (Mar. 1994) 35:518 (Abstract 3088).
Appendix B (item 4): G. Granger et al. "Basic and clinical studies of intralesional therapy of gliomas with allogeneic lymphoid cells" Proc. Amer. Assoc. Cancer Res. (Mar. 1995) 36:472 (Abstract 2812).
Appendix D: List of patients treated according to the invention up to Sep. 26, 1996.
Bishara et al., "A short human and mouse MLR assay utilizing lymphokine (Il-2 and IL-3) secretion as an early activation event," Transplantation 51:1104-1109 (1991).
Danzer et al., "Interferon-gamma plays a key role in the human mixed lymphocyte culture," Bone Marrow Transpl. 18:991-996 (1996).
Danzer et al., "Cytokine interactions in human mixed lymphocyte culture," Transplantation 57:1638-1642 (1994).
Grabstein et al., "Expression of interleukin 2, interferon-.gamma., and the IL 2 receptor by human peripheral blood lymphocytes," J. Immunol. 136:4503-4508 (1986).
Matthews and Sullivan, "A role for tumor necrosis factor-.alpha. in the human mixed lymphocyte culture reaction," Immunol. Cell Biol. 70:107-110 (1992).
Novelli et al., "Modulation of interferon-.gamma. receptor during human T lymphocyte alloactivation," Eur. J. Immunol. 23:1226-1231 (1993).
Novelli et al., "Blockade of physiologically secreted IFN-.gamma. inhibits human T lymphocyte and natural killer cell activation," J. Immunol. 147:1445-1452 (1991).
Pawelec et al., "Differential secretion of tumor necrosis factors-.alpha. and granulocyte/macrophage colony-stimulating factors but not interferon-.gamma. from CD4.sup.+ compared to CD8.sup.+ human T cell clones," Eur. J. Immunol. 19:197-200 (1989).
Shalaby et al., "The involvement of human tumor necrosis factors-.alpha. and -.beta. in the mixed lymphocyte reaction," J. Immunol. 141:499-503 (1988).
Declaration by John C. Hiserodt regarding Interpretation of M.R.I. Data.
Redd et al., "Allogeneic tumor-specific cytotoxic T lymphocytes" Cancer Immunol. Immunother. (1992) 34:349-354.
Schlitz et al., "Treatment of 9L gliosarcoma with interferon-gamma enhances its cytolysis by alloreactive cytotoxic T lymphocytes in vitro" FASEB J. (1995) 9(4):A1044 (Abstract 6052).
Albright et al., "Immunogenetic control of brain tumor growth in rats" Cancer Res. (1977) 37:2512-2521.
Barba et al., "Intratumoral LAK cell and interleukin-2 therapy of human gliomas" J. Neurosurg. (1989) 70:175-182.
Bellgrau, "Induction of cytotoxic T cell precursors in vivo" J. Exp. Med. (1983) 157:1505-1515.
Carson et al., "Rat mitogen-stimulated lymphokine activated T killer cells: Production and effects on C.sub.6 glioma cells in vitro and in vivo in the brain of wistar rats" J. Immunother. (1991) 10(2):131-139.
Cavallo et al., "Role of neutrophils and CD+ T lymphocytes in the primary and memory response to nonimmunogenic murine mammary adenocarcinoma made immunogenic by IL-2 gene" J. Immunol. (1992) 149(11):3627-3635.
Chang et al., "Phase I clinical trial of allogeneic mixed lymphocyte culture (cytoimplant) delivered by endoscopic ultrasound (EUS)-guided fine needle injection (FNI) in patients with advanced pancreatic carcinoma" Gastroenterology (1997) 112(4):A546.
Colombo et al., "Tumor cells engineered to produce cytokines or cofactors as cellular vaccines: Do animal studies really support clinical trials?" Cancer Immunol. Immunother. (1995) 41:265-270.
Finke et al., "Characterization of the cytolytic activity of CD+ tumor-infiltating lymphocytes in human renal carcinoma" Cancer Res. (1990) 50:2363-2370.
Fleshner et al., "Potential of allogeneic tumoricidal cytotoxic T lymphocytes in brain tumor adoptive immunotherapy" J. Cell. Biochem. (1990) Suppl. 0 (14 part B):95 (abstract CE 407).
Fletcher et al., "Recent advances in the understanding of the biochemistry and clinical pharmacology of interleukin-2" Lymphokine Research (1987) 6(1):45-57.
Hayes et al., "Recombinant interleukin-2-related intracebral toxicity and LAK/rIL-2 therapy for brain tumors" Lymphokine Res. (1988) 7(3):337 (Abstract 9.25).
Jeffes III et al., "Therapy of recurrent high grade gliomas with surgery, autologous mitogen-activated IL-2-stimulated (MAK) killer lymphocytes, and rIL-2: II. Correlation of survival with MAK cell tumor necrosis factor production in vitro" Lymphokine Cytokine Res. (1991) 10(2):89-94.
Kondo et al., "Rationale for a novel immunotherapy of cancer with allogeneic lymphocyte infusion" Med. Hypotheses (1984) 15:241-277.
Kruse et al., "Analysis of interleukin-2 and various effector cell populations in adoptive immunotherapy of 9L rat gliosarcoma: Allogeneic cytotoxic T lymphocytes prvent tumor take" Proc. Natl. Acad. Sci. USA (1990) 87:9577-9581.
Kruse et al., "Artificial-capillary-system development of human alloreactive cytotoxic T-lymphocytes that lyse brain tumour" Biotechnol. Appl. Biochem. (1997) 25: 1-9. Galley proof of article that was later published as Biotechnol. Appl. Biochem. (1997) 25:197-205.
Kruse et al., "Cellular therapy of brain tumors: Clinical trials" Advances in Neuro-Oncology II, Kornblith et al., eds., Future Publishing Company, Inc., Armonk, NY, (1997) Chapter 22, pp. 487-504.
Kruse et al., "Development of human allogeneic CTL in an aritificial capillary system for intercavitary treatment of malignant glioma" Proc. Amer. Assoc. Cancer Res. (1995) 36:474 (abstract No. 2822).
Kruse et al., "Immune therapy of recurrent mailgnant gliomas: Intracavitary allogeneic cytotoxic T lymphocytes and human recombinant interleukin-2" FASEB J. (1996) 10(6):A1413 (abstract 2387).
Kruse et al., "Intracranial administrations of single or multiple source allogeneic cytotoxic T lymphocytes: chronic therapy for primary brain tumors" J. Neuro-Oncol. (1994) 19:161-168.
Kruse et al., "Migration of activated lymphocytes when adoptively transferred into cannulated rat brain" J. Neuroimmunol.(1994) 55:11-21.
Kruse et al., "Systemic chemotherapy combined with local adoptive immunotherapy cures rats bearing 9L gliosarcoma" J. Neuro-Oncol. (1983) 15:97-112.
Leshem et al., "In vitro elicitation of cytotoxic response against a nonimmunogenic murine tumor be allosensitization" Cancer Immunol. Immunother. (1984) 17:117-123.
Merchant et al., "Adoptive immunotherapy for recurrent gliobastoma multiforme using lymphokine activated killer cells and recombinant interleukin-2" Cancer (1988) 62:665-671.
Merchant et al., "Immunotherapy for malignant glioma using human recombinant interleukin-2 and activated autologous lymphocytes. A review of pre-clinical and clinical investigations" J. Neuro-Oncol. (1990) 8:173-188.
Mitchell et al., "Active specific immunotherapy of melanoma with allogeneic cell lysates. Rationale, results, and possible mechanisms of action" Ann. N.Y. Acad. Sci. (1993) 690:153-166.
Naganuma et al., "Complete remission of recurrent glioblastoma multiforme following local infusions of lymphokine activated killer cells. Case report" Acta. Neurochir. (1989) 99:157-160.
Rosenberg et al., "A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high doses interleukin-2 alone" New England Journal of Medicine (1987) 316(15):889-897.
Schlitz et al., "Movement of allogeneic cytotoxic T lymphocytes (aCTL) infused into the parietal region of 9L gliosarcoma bearing brain" Proc. Amer. Assoc. Cancer Res. (1995) 36:458 (abstract No. 2727).
Strausser et al., "Lysis of human solid tumors by autologous cells sensitized in vitro to alloantigens" J. Immunol. (1981) 127(1):266-271.
Strelein et al., "Unraveling immune response" Science (1995) 270:1158-1159.
Topalian et al., "Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: A pilot study" J. Clin. Oncol. (1988) 6(5):839-853.
Yoshida et al., "Local administration of autologous lymphokine-activated keller cells and recombinant interleukin-2 to patients with malignant brain tumors" Cancer Res. (1988) 48:5011-5016.
Zarling et al., "Generation of cytotoxic T lymphocytes to autologous human leukemia cells by sensitization to pooled allogeneic normal cells" Nature (1978) 274:269-271.
Storniolo et al., Cander 15:1261-8, 1999. "An investigational new drug treatment program with gemcitabine: results for over 3000 patients with pancreatic cancer." (Complete article).
Hildalgo et al., J. Clin. Oncol. 17:585-92, 1999. "Phase I-II study of gemcitabine and fluorouracil as a continuous infusion in patients with pancreatic cancer" (Abstract only).
Dugan et al., Pancreas 17:325-33, 1998. "Current concepts in pancreatic cancer: Symposium summary" (Abstract only).
Stephens, Oncol. Nurs. Forum 25:87-93, 1998. "Gemcitabine: A new appraoch to treating pancreatic cancer" (Abstract only).
Carmichael, Digestion 58:503-7, 1997. "Clinical response benefit in patients with advanced pancreatic cancer. Role of gemcitabine" (Abstract only).
Burris et al., Eur. J. Cancer 33:S18-22, 1997. "Assessing clinical benefit in the tratment of pancreas cancer: Gemcitabine compaed to 5-fluorouracil" (Abstract only).
Rotherberg et al., Ann. Oncol. 7:347-53, 1996. "A phase II trial of gemcitabine in patients with 5-FU-refractory pancreas cancer" (Abstract only).
Carmichael et al., Br. J. Cancer 73:101-5, 1996. "Phase II study of gemcitabine in patients with advanced pancreatic cancer" (Abstract only).
Casper et al., Invest. New Drugs 12:29-34, 1994, "Phase II trial of gemcitabine (2,2'-difluorodeoxycytidine) in patients with adenocarcinoma of the pancreas" (Abstract only).
Burris et al., J. Clin. Oncol. 15:2403-13, 1997. "Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial" (Abstract only).
Michael et al., Oncology (Huntingt) 11:1615-22, 1625-7, 1997. "Clinical experience with gemcitabine in pancreatic carcinoma" (Abstract only).

Abstract

Description