COMPOSITIONS FOR ENHANCING ADCC THERAPY

Abstract: A pharmaceutical composition consisting of two locally separated preparations is provided which selectively suppresses tumor cell growth. Use of the composition consists of co-administering M-CSF and/or GM-CSF with specified isotypes of monoclonal antibodies.

Description: .-COMPOSITIONS FOR ENHANCING ADCC THERAPY The present invention relates to pharmaceutica compositions and the use thereof for selectively suppressin tumor cell growth in a human patient and specifically to th conjoint use of M-CSF and/or GM-CSF with certain monoclona antibodies to destroy tumor cells. The recognition and use of antibody dependent cel cytotoxicity (ADCC) reaction has been employed in developin cancer therapies, by using the antibodies alone or by using th antibodies to direct purported chemotherapeutic agents to th site of the cancer. See, e.g., published European Paten Application 194,851 which discloses treating a patient with beta 1,3-glucan lentinan followed by administration of anti-tumo monoclonal antibodies. PCT application no. O87/00054 disclose attaching monoclonal antibodies to effector cells primed by gamm interferon and/or IL-2 and re-injecting a patient with th combination. For general information on the ADCC reaction, se also, A. F. Lopez et al, J. Immunol.. 131(6) :2983-2988 (1983); M. A. Vadas et al, J. Immunol., 130(2) .795-799 (1983); T. J. Kipp et al, J. EXP. Med. , 161:1-17 (1985). Similar combination therapies were contemplated by Houghto et al., who speculated that "interferons, interleukin-2, tumo necrosis factor and other lymphokines could enhance the activit of effector cells directed to the tumor by MAbs." [Houghton, A.N., & D.A. Scheinberg, Sem. Oncol., 13: 165-179 (1984)]. Notwithstanding this generalized optimism, many of these factors to which Houghton et al. refer have in fact proven ineffective at augmenting immune-mediated tumor destruction. THE INVENTION Applicants have surprisingly found that M-CSF and GM-CSF are effective in potentiating the σytolysis of tumor cells by monocytes in an ADCC protocol. Specifically, M-CSF and GM- CSF are formulated into pharmaceutical compositions which prime macrophages such that they become capable of selectively lysing tumor cells in vivo . The compositions consist of two preparations which are locally separated. One such preparation is the colony stimulating factor ("the CSF") M-CSF and/or GM-CSF. The other preparation is a monoclonal antibody directed to an antigen associated with the tumor cells sought to be lysed. The present invention further contemplates the use of M-CSF and GM-CSF for priming macrophages either in vivo or ex vivo. The preferred embodiment of this invention utilizes as the colony stimulating factor, M-CSF, also known as CSF-l, monocyte colony stimulating factor, and macrophage colony stimulating factor. M-CSF induces colonies which contain primarily macrophages . It has been described in Wong et al . , Science, 235:1504-1508 (1987) and publications by E. R. Stanley. Its production by recombinant DNA techniques is described in U.S. application Serial No. 860,377. A truncated version is described in PCT/US86/00238. Another CSF within the scope of this invention is GM-CSF, also known as granulocyte- acrophage colony stimulating factor. It is described in detail in Wong et al., Science , 2_2_8_: 810-815 (1985) and references cited therein. Wong et al . also teach its production via recombinant DNA techniques. While GM-CSF had been recognized as exhibiting significant .in vitro activity on the various hematopoietic progenitor cells, its use in treatment of clinical conditions remains prospective. See also, EPO Pub. No. 0,183,350 and PCT Application WO87/02060. M-CSF displays surprising efficacy over other lymphokines affecting monocytes and macrophages in ADCC experiments in priming those cells to lyse target tumor cells. The term "priming" or "primed" macrophage in this context refers to the effect on a responsive macrophage of exposure to a lymphokine. The macrophage becomes primed, and is therefore capable of becoming fully cytolytic (or activated) upon exposure to a monoclonal antibody. [See, e.g., D. Adams, Ann. Rev. Immunol. (1984).] It has been found that M-CSF can prime macrophages to kill tumor cells in a significantly shorter time period than other immune regulators, and the effector cells require less monoclonal antibody to achieve such kill. Similarly, GM-CSF also shows efficacious results in such a protocol. A preparation consisting of a monoclonal antibody directed to antigens associated with the tumor cells to be lysed (e.g., a cell surface antigen) is another essential component of this invention. This monoclonal antibody may be selected from; murine antibody isotypes IgG3, IgG2A, IgG2B, and human antibody isotype IgGl. One aspect of the present invention provides a method for selectively killing tumor cells in a human patient by priming macrophages pre-incubated with M-CSF and/or GM-CSF and co-administering the primed cells with the above- identified monoclonal antibodies to tumor-associated antigens to the patient. In this method the macrophage priming occurs ex vivo. Another aspect of the invention provides a method for selectively killing tumor cells in a human patient without necessitating extracorporeal treatment. This is accomplished by directly co-administering to the patient an effective dose of M-CSF and/or GM-CSF and an effective dose of a monoclonal antibody selected from among the above isotypes. In this method, the macrophage priming occurs jln vivo. The M-CSF and GM-CSF employed in the present invention are desirably purified human proteins, free from association with other contaminating proteins. The M-CSF and GM-CSF so employed include the natural proteins, recombinant versions thereof, and derivatives and analogs thereof, which may contain a ino acid deletions, substitutions and/or insertions, but which retain the characteristic biological activity and are encoded by cDNAs capable of hybridizing to cDNAs for the naturally occurring versions. Also included are naturally-occurring isotypes or allelic variations in the protein or its coding sequence resulting from expression of the protein in different members of a species. Several specific monoclonal antibodies of the human isotype IgGl and the murine antibody isotypes IgG2 and IgG3 are described in EPA 194,851. The type of tumor cells which are destroyed by this -method depends on the particular antigen to which the monoclonal antibodies are directed. For example, co-administration of M-CSF and/or GM-CSF with a murine IgG3 antibody developed against human lung carcinoma cells directs the primed macrophages to localize around, and thus lyse, those lung tumor cells. Similar antibodies of the murine isotypes IgG2A and IgG2B or human isotype IgGl directed to other tumor types, such as breast, colon and liver, are available, or can be developed by one of skill in the art. Examples of existing monoclonal antibodies which are potentially useful according to the method include: PM4E9917, a murine IgG2a directed against a liver carcinoma cell surface antigen [R. Carlson et al., J. Clin. Invest.. 2j5:40-46 (1985)]; 791T/36, a murine IgG2b directed against a colon carcinoma cell surface antigen [L.G. Durrant, Cancer Res.. 4.6:3543-3549 (1986)]; and 083-17-1A, a murine IgG2a directed against a colon carcinoma cell surface antigen [H. Koprowski et al, Somat. Cell Genet.. 5_:957-972 (1979)]. As used herein, the term "co-administration" encompasses serial or sequential administration of the components of the method, including pre-administration of one or more components in favor of the others. For example, in one embodiment of the invention, the monoclonal antibody is administered to the patient as a first step and subsequently M-CSF and/or GM-CSF is administered. In a second embodiment, the CSF is first administered, and subsequently a monoclonal antibody and the other CSF are administered. In a third embodiment, the CSF is administered prior to the administration of the monoclonal antibody. In a fourth embodiment the monoclonal antibody and the CSF are administered simultaneously. Thus, when antibody and M-CSF and/or GM-CSF are administered separately to the patient, the CSF causes in vivo priming of the macrophages in the body and the antibody becomes associated with the tumor cells. The primed macrophages then specifically recognize tumor cell/antibody complex. Alternatively, M-CSF and GM-CSF may be used to prime monocytes .in. vitro, which are then readministered to the patient who has been pretreated with antibody and the same specific cytolysis may occur. In this regimen, antibody may also be given after re-introduction of the in vitro priming of monocytes by CSF. In yet another embodiment, M-CSF and/or GM-CSF are used to prime the monocytes in vitro by incubation with the CSF and then subsequently "armed" in vitro by attachment to the antibody. Attachment of the primed macrophages to the monoclonal antibody ("arming") .in vitro may be accomplished in a number of conventional ways. [See, e.g., Z. Steplewski et al. Science. 221:865-867 (1983)]. The complex is then re- introduced into a patient to obtain the same specific tumor cell cytolysis. Other orders of administration are also encompassed by this term. The use of these compositions may find particular application as a therapy to control metastatic spread of tumor cells following surgical removal of a primary tumor. As well as the components enumerated specifically herein, this method may utilize other substances that are active on tumor-associated monoclonal antibodies. The components of this method can be systemically administered parenterally. Alternatively, these components may be administered intravenously. Where desired for treatment, it may also be possible to administer one or more components of the method subcutaneously. When systemically administered, the therapeutic preparations of the monoclonal antibody and CSF and optional second CSF for use in this invention are in the form of a pyrogen-free, parenterally acceptable aqueous solutions. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The dosage regimen involved in the use of these compositions for suppressing susceptible tumors will be determined by the attending physician considering various factors which modify the action of drugs, e.g., the condition, body weight, sex, and diet of the patient, the severity of any infection, time of administration and other clinical factors. The CSF component should be administered in a dosage insufficient to cause a systemic toxic reaction, but sufficient to elicit the potentiating response on the effector cells. Generally, the daily regimen should be in the range of 200-1000 micrograms of CSF polypeptide, or 50 to -5000 units (ie, a unit being the concentration of polypeptide which elicits a half maximal response in an in vitro colony forming assay or a macrophage activation assay) . The dosage cited above may be adjusted to compensate for whether the method involves the use of only one CSF or a combination of CSFs. The effective dosages of the monoclonal antibody are in the range of 0.1 to 1 mg/kg body weight daily, depending on the physician's estimate of tumor size and the extent of metastasis. Progress of the treated patient can be monitored by periodic assessment of the metastasis. The following example illustrates the significant advantages in the employment of M-CSF in ADCC reactions according to the present invention. EXAMPLE I Peripheral blood monocytes are prepared by adherence to tissue culture plates by conventional methods. Approximately 5 X 10 5 to 5 X 10 6 adhered monocytes/well are incubated at 37°C for either 24, 48 or 72 hours in 20 ng recombinant M-CSF per tissue culture medium,as verified by amino acid analysis. Control plates of monocytes were incubated under the same conditions and times but without the addition of M-CSF. The monocytes from each plate are added to an ADCC protocol, performed as described in Herlyn et al, supra. employing monoclonal antibodies of the murine isotype IgG3 prepared against colon carcinoma surface antigens and colon carcinoma cell line, LS-174 [NeoRx Inc.]. The colon carcinoma cells employed as the target cells in the ADCC protocol are prelabelled with [3]H thymidine and all cell death is measured by release of [3]H thymidine. The ADCC reaction was allowed to run for 5 days. Fig. 1 shows the results of the ADCC reaction by plotting days in ADCC vs. percentage of target cell lysis. The target to effector cell ratio in the experiment of Fig 1 is 1:100; however, the same results can be obtained with a T:E ratio of 1:10. The antibody concentration in the experiment of Fig. 1 is 100 ng/ml. The adhered monocytes incubated in M-CSF clearly caused more target cell death at all pre-incubation times than did the controls. In general, M-CSF pretreated monocytes kill approximately two and one- half times as many target cells as untreated monocytes. The adhered monocytes which were preincubated in M-CSF for 72 hours allowed the fastest rate of killing to occur in the ADCC reaction, achieving maximum killing within 24 hours. Fig. 2 demonstrates that monocytes pretreated with M-CSF for 72 hours required 10-fold less antibody to acheive target cell killing than did untreated monocytes in the ADCC protocol employing a T:E ratio of 1:100. Similar results were demonstrated with T:E: ratios as low as 1:10.

Claims: WHAT IS CLAIMED IS: 1. A pharmaceutical composition for selectively lysing tumor cells in a human patient characterized in that the composition consists of two preparations which are locally separated and comprising: (a) a monoclonal antibody directed to an antigen associated with said tumor cells and selected from the group consisting of murine antibody isotypes IgG3, IgG2A and IgG2B and human antibody isotype IgGl; and (b) a colony stimulating factor selected from the group consisting of M-CSF and GM-CSF. 2. The composition of claim 1 in which the colony stimulating factor is M-CSF. 3. The composition of claim 1 in which the colony stimulating factor is GM-CSF. 4. The composition according to claim 1 wherein the composition contains both M-CSF and GM-CSF. 5. Use of (a) a monoclonal antibody directed to an antigen associated with tumor cells and selected from the group consisting of murine antibody isotypes IgG3, IgG2A and IgG2B, and human antibody isotype IgGl; and (b) a colony stimulating factor selected from the group consisting of M-CSF and GM-CSF for the preparation of a pharmaceutical product comprising a combination of the monoclonal antibody and the colony stimulating factor suitable for separate sequential use of the monoclonal antibody and the colony stimulating factor in the treatment of cancer. 6. The use of claim 5 wherein the colony stimulating factor is M-CSF. 7. The use of claim 5 wherein the colony stimulating factor is GM-CSF.

Inventors: DE VILLIERS JEAN, MUFSON R ALLAN, DE VILLIERS, JEAN, MUFSON, R., ALLAN
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1988
Main classification: A61K37
IPC codes: A61K38, A61K39, A61P35