WO1996040176A1

WO1996040176A1 - Therapeutic uses of monoclonal antibody ta99 in combination with interleukin-2 and/or lymphokine activated killer cells

Info

Publication number: WO1996040176A1
Application number: PCT/US1996/009605
Authority: WO
Inventors: Alan N. Houghton; Neil Goldstein
Original assignee: Sloan-Kettering Institute For Cancer Research; Imclone Systems Incorporated
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19
Also published as: AU6104396A

Abstract

This invention provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2 and a pharmaceutically acceptable carrier. This invention further provides the above composition further comprising lymphokine activated killer cells.

Description

THERAPEUTIC USES OF MONOCLONAL ANTIBODY TA99 IN COMBINATION WITH INTERLEUKIN-2 AND/OR LYMPHOKINE ACTIVATED KILLER CELLS

Background of the Invention

There is both laboratory and clinical evidence to suggest that immunotherapy may play a role in the treatment of malignant melanoma. For example, T-cell infiltration into melanoma lesions is often associated with regression of primary melanoma and vitiligo, an autoimmune phenomenon involving localized or systemic pigmentation, has been suggested to be a good prognostic indicator for patients with metastatic melanoma. In addition, various melanoma-associated antigens have been defined by both monoclonal antibodies and T-cell reactivity. These include assorted gangliosides (e.g., GD2, GD3) , the high molecular weight melanoma associated antigen (HM -MAA) , MAGE 1-3, MART-1, tyrosinase and gP75. Immunotherapeutics, based on a several of these antigens have been utilized in a number of clinical studies with varying results .

Interestingly, many of these antigens are intracellular in origin yet animals and humans appear to be able to generate immune responses to these proteins. gP75 (also called pigmentation associated antigen and tyrosinase related protein-1 or TRP-1) is included in this group. This antigen is a highly glycosylated protein associated with the melanosomes. In the mouse, gP75 has been mapped to the brown locus and is associated with coat color. There is about 90% homology between murine and human gP75 and 43% homology between gP75 and tyrosine. Although originally described as a "tyrosinase", the function of gP75 has not been fully elucidated. TA99 is a mouse monoclonal antibody (IgG2a) recognizing an epitope on gP75. The antibody was generated following the immunization of mice with the highly pigmented human melanoma SK-MEL-23 and fusion to NS1 cells. TA99 was found to be specific for pigmented cells and, in spite of its intracellular distribution, has been shown to localize to human melanoma xenografts in athymic nude mice. Because of the intensity of the localization in animal models, it was hypothesized that TA99 may show utility as an immunotherapeutic reagent. Applicants began to address this question by treating C57BI/6 mice carrying the highly aggressive B16 melanoma tumor with TA99. Results indicate that the antibody is effective in retarding the growth of B16F1 and increasing survival of the tumor bearing animals. These studies suggest that strategies can be defined that will allow the use of TA99 in the immunotherapy of melanoma.

Summary of the Invention

This invention provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2 and a pharmaceutically acceptable carrier. This invention further provides the above composition, further comprising stimulated splenocytes from an appropriate host. The stimulated cells may be peripheral mononuclear blood cells, lymph node cells or lymphokine activated killer cells.

This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2, further comprising appropriate natural killer cells which are stimulated and a pharmaceutically acceptable carrier.

This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of stimulated splenocytes from an appropriate host and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of appropriate natural killer cells which are stimulated and a pharmaceutically acceptable carrier. The stimulated cells may be peripheral mononuclear blood cells, lymph node cells or lymphokine activated killer cells.

Finally, this invention provides a method of treating melanoma which comprises administering effective amount of the above described pharmaceutical compositions Brief Description of the Figures

Figure 1 : Comparison of TA99 VS. an Irrelevant Isotvpe Matched Control on the Growth of B16F1 Cells in C57BL/6 Mice

Mice were inoculated with B16F1 (50000 subcutaneously) on day zero. Antibody injections (1 mg/injection) were begun at the same time. On day zero, animals were given 2 separate intraperitoneal injections followed by 8 other inoculations over 3 weeks (total antibody injected = 10 mg/animal) . Tumor measurements were done over the course of the study (36 days) and volumes defined as described in Materials and Methods. IA:Animals treated with irrelevant isotype matched controls. IB: Animals treated with TA99.

Figure 2 : Comparison of Intact TA99 VS. F(ab')2 in the on the Growth of BlβFl Cells in C57BL/6 Mice

Mice were inoculated with B16F1 cells as described. Beginning on day zero, animals received PBS, intact TA99, or an F(ab')2 fragment of this antibody as described in Materials and Methods. 2A: PBS control.

2B: Intact TA99. 2C: F(ab')2 fragment.

Figure 3 : The Effect of Coadministration of TA99 with IL-2 and IL-2 Stimulated Splenocytes (24 Hours) on the Growth of B16F1 Cells in C57BL/6 Mice

Splenocytes, isolated from C57B1/6 mice were stimulated in vitro with IL-2 (10 ng/ml) for 24 hours prior to iv injection into C57/B16 mice on the same day in which B16F1 cells were implanted into the animals. Controls and antibody treatments began on the same day. Animals received a total of 8 mg of TA99 and 200 U/day of IL-2 over 3 weeks. 3A-3D: Results of the individual treatments on the growth of the B16F1 tumor. 3E: Survival data for the individual treatments.

Figure 4 : The Effect of Coadministration of TA99 with IL-2 and IL-2 Stimulated Splenocytes

(72 Hours) on the Growth of B16F1 Cells in C57BL/6 Mice.

Splenocytes were isolated and stimulated in vitro with IL-2 for 72 hours prior to iv injection into C57/B16 mice with TA99 as described in Materials and Methods

Treatment began on the same day as tumor implantation. Animals received a total of 8 mg of TA99 and 200 U/day of IL-2 over 3 weeks. 4A-4D: Results of the individual treatments on the growth of the B16F1 tumor. 4E: Survival data for the individual treatments .

Figure 5 : Effect of TA99 (Lower Dose/Increased Treatment Time) on the Growth of B16F1 Tumors in C57BL/6 Mice.

Animals were implanted with B16F1 tumors 5 as described in Materials and Methods.

Treatments began on day 5 post-tumor implantation and consisted of lower dose TA99 (500 ug/injection) for a longer period of time (2 injections per week for

10 5 weeks) . 5A-5B: Tumor growth of B16F1 subcutaneous tumors in C57BL/6 mice following treatment with TA99 (5mg over 5 weeks) beginning on Day 5. 5C: Tumor incidence in mice transplanted with B16F1

15 following treatment with TA99 (4.5mg over

5 weeks) beginning 5 days post- transplantation.

Detailed Description of the Invention

This invention provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2 and a pharmaceutically acceptable carrier. This invention further provides the above composition, further comprising stimulated splenocytes from an appropriate host. In an embodiment, the splenocytes are stimulated by interleukin-2. In a separate embodiment, the stimulated cell is peripheral blood mononuclear cell (PBMC) . In a further embodiment, the PBMC is human PBMC. In a still further embodiment, the stimulated cell is lymph node cells. These cells may be stimulated by inteleukin-2. In a separate embodiment, the cells are lymphokine activated killer (LAK) cells. And in a further embodiment, the LAK cells are activated by interleukin-2.

For the purposes of this invention "pharmaceutically acceptable carriers" means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

An "effective amount" of the pharmaceutical composition is any amount of the pharmaceutical composition effective to inhibit the proliferation of tumor cells or neoplastic cells. Methods of determining an "effective amount" are well known to those skilled in the art and depend upon factors including, but not limited to: the size of the patient and the carrier used.

This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2, further comprising appropriate natural killer cells which are stimulated and a pharmaceutically acceptable carrier. In an embodiment, the natural killer cells are stimulated by interleukin-2. In a separate embodiment, the stimulated cells are peripheral blood mononuclear cells (PBMC) . In a further embodiment, the PBMC are human PBMC. In a still further embodiment, the stimulated cells are lymph node cells. In another embodiment, the lymph node cells are human lymph node cells. These cells may be stimulated by inteleukin-2. In a separate embodiment, the cells are lymphokine activated killer (LAK) cells. And in a further embodiment, the LAK cells are activated by interleukin-2.

This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of stimulated splenocytes from an appropriate host and a pharmaceutically acceptable carrier. In an embodiment, the splenocytes are stimulated by interleukin-2.

This invention also provides a pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of appropriate natural killer cells which are stimulated and a pharmaceutically acceptable carrier. In an embodiment, the splenocytes are stimulated by interleukin-2.

Finally, this invention provides a method of treating melanoma in a subject which comprises administering effective amount of the above described pharmaceutical compositions to the subjects. In an embodiment, the subject is a patient having melanoma.

"Administering" means any of the standard methods of administering a pharmaceutical composition known to those skilled in the art. Examples include, but are not limited to intravenous, intramuscular or intraperitoneal administration.

This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details Materials and Methods

Cell Lines and Medium:

B16F1 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine (Hyclone) serum, 2mM L-glutamine (Sigma) , and antibiotics. Recombinant mouse IL-2 was obtained from Genzyme (Boston MA) and stored at -70C following reconstitution.

Animals :

C57B1/6 mice were obtained from Charles River Laboratories at 5-6 weeks of age and were fed and watered ad libi tum. All studies were done with animals 8-10 weeks old.

Animal Studies : The B16F1 melanoma grown in syngeneic C57B1/6 mice was used as applicants' model to study the effects of TA99 on tumor growth and progression in vivo . In brief, 50000 B16F1 cells were implanted subcutaneously into the animals. Tumors develop over a 10-14 day period and measurements are taken a minimum of 3 times weekly, tumor volume (mm³) was computed as 7r/6 x a(b) ² were a>b. In addition, the survival of animals within each treatment group was also determined.

Initially, the effect of TA99 on the growth of B16F1 in C57B1/6 mice was compared to an irrelevant isotype matched control monoclonal antibody. These studies revealed that irrelevant monoclonal antibodies had no effect on tumor progression. On the basis of these results, PBS (phosphate buffered saline) was used as the control in subsequent experiments.

The dose schedule for the animals experiments is briefly described. In the first series of studies, TA99 was injected twice on day zero (1 mg/injection for a total of

2mg at the time of tumor implantation) and 3 times per week (1 mg/injection) over the next 21 days (total injection dose = 10 mg per animal) . In latter studies, the concentration per injection of TA99 was decreased

(500 ug/injection) with a concomitant increase in the overall number of inoculations (2 times per week for 5 weeks for a total injected dose of 5 mg) .

Generation of Antibody Fragments and Animal Studies:

The generation of F(ab')2 fragments of TA99 was done using reagents available from Pierce. The success of fragmentation was determined using SDS PAGE.

Animals were inoculated with B16F1 cells as described above. Antibody treatments began on day zero and continued for three weeks. Mice were injected with a total of 10 mg of intact antibody and a molar equivalent of the F(ab')2 fragment. Control animals received sham injections of PBS. Tumor volumes were determined as described above.

In Vivo Studies with IL-2 Stimulated Splenocytes: Splenocytes from C57B1/6 mice were aseptically collected and washed two times with pre-warmed serum free DMEM. Cells were plated at 2 x 10⁶ in complete medium containing 10 ng/ml of recombinant mouse IL-2 and the cultures maintained for either 1 or 3 days. Viable cells were harvested and injected intravenously into the test animals at 1-2 x 10⁷ cells per animal on the same day as tumor was implanted (day zero) or 5 days post-tumor implantation (day 5) . The groups consisted of PBS control, IL-2 stimulated splenocytes alone, TA99, and TA99 plus IL-2 stimulated splenocytes. All treatments followed the injection of the IL-2 stimulated splenocytes. During the treatment regimen, all animals received daily (5 per week) intraperitoneal injections of IL-2 (100 ng/animal/day) .

Experimental Results and Discussion

TA99 is a murine monoclonal antibody (lgG2A) , generated against human melanoma that reacts equally with both murine and human gP75. Studies with human tumor xenografts in athymic nude mice have shown that the monoclonal antibody rapidly localizes to the growing tumors. This was a surprising observation since gP75 is an intracellular antigen associated with the melanosomes and not thought to be expressed on the cell surface. The most likely explanation for these results is the high concentration of gP75 within the tumor mass as a result of necrosis and the subsequent accretion of radiolabelled TA99.

Based on these results, it was decided to determine if the localization of TA99 to melanomas is accompanied by a therapeutic effect. In order to test the validity of this hypothesis, TA99 was screened using an autologous tumor model consisting of grafted B16 (gp75 positive) in C57B1/6 mice. A number of parameters were assayed including dose response and timing of TA99 inoculations and the effect of IL-2 stimulated LAK cells. The latter variable was included because it has been shown in cell depletion assays that NK and CD4+ T cell are necessary for the TA99 inhibitory effect (unpublished data) .

Figure 1 shows the results of a representative study in which the effect of TA99 on the growth of B16F1 cells in C57/B16 mice (Figure IB) is compared to an isotype matched irrelevant monoclonal antibody (Figure IA) . As can be seen, there is a significant delay of tumor onset in the control group when compared to animals treated with TA99. In addition, all of the control animals are dead by day 30 whereas 4/5 animals treated with TA99 were alive when the study was terminated on day 36.

Applicants were interested in determining if the inhibitory effect of TA99 was due direct binding of the antibody to gP75 or if the Fc was needed for secondary effector function. For this reason, the effect of intact TA99 was compared with its bivalent F(ab')2 fragment at molar equivalence. The results of this study (Figure 2) show that in vivo growth of B16F1 cells is not effected by the addition of F(ab')2 fragments. As can be seen, animals in the control and F(ab')2 groups develop larger tumors and 4/5 are dead by day 30. On the other hand, the animals inoculated with intact TA99 have smaller tumors and 3/5 are alive at the termination of the study (day 35) . These data suggest that the Fc region in an important component of the tumor inhibitory effect of TA99.

Results of cell depletion studies have indicated that both CD4+ and NK cells but not CD8+ T cells are necessary for the inhibitory effect in vivo (A. Houghten, unpublished data) . These data suggest that effector cells play a role in the inhibition of tumor growth by TA99. To address this phenomenon, applicants ran a number of studies in which IL-2 stimulated syngeneic splenocytes were co-administered with TA99 to the animals at various times post-tumor implantation. Figure 3 shows the results of a study in which the animals received 10⁷ IL-2 activated splenocytes (1 day in culture) , TA99, or a combination of both starting at the time of tumor implantation (day zero) . In addition, all animals received injections of IL-2 (200 U per animal) 5 times weekly. As can be seen in Figure 3A, all of the PBS animals and 4/5 from IL-2/cell alone group developed tumors by day 27. Animals in the TA99 IL-2 group develop tumors over an extended period of time period (day 22 to day 42) . In addition, 1/5 animals remained tumor free over the course of the study (78 days) and continued to be disease-free for the 6 month followup. More interestingly, animals treated with the combination of TA99, IL-2, and activated splenocytes show a dramatic inhibition of tumor growth with 2/5 developing tumors by day 35, 1/5 developing a tumor very late in the study (day 57) , and 2/5 remaining disease-free during the study and during the six month followup. Animal survival data is shown in Figure 3B and correlates with the tumor progression results; i.e., animals treated with the combination of TA99, IL-2, and activated splenocytes show increased long term survival over controls and TA99 alone.

Figure 4 shows the effects of coadministration of TA99 and IL-2 activated splenocytes (3 days in culture) to animals 5 days post-tumor implantation. In these studies, tumors quickly developed in the PBS and IL-2 stimulated cells alone groups. Although both of the test groups (TA99 and TA99 + cells) show inhibition of tumor development, there appears to be very little difference between the groups in either the appearance and progression of tumors and a slight survival advantage during the immunotherapy stage (Figure 4B; note: there is a 5 day difference in median [50%] survival between TA99 alone [day 36] and the combination [day 41] ) . These data indicate that TA99 is effective in inhibiting tumor growth and progression at an early stage of tumor development (i.e., within 5 days of tumor inoculation) since it has been observed that the antibody has no effect on 7 day old tumors (unpublished observations) . In addition, the data also suggest that there exists a therapeutic window in which coadministration of IL-2 activated killer cells is more effective than antibody alone.

In the previous studies, it was determined that treatment of animals with TA99 by day 5 was as effective as beginning the protocol at the time of tumor implantation. In addition to the question to tumor susceptibility, it was also important to define a minimum effective dose for

TA99 as well as the optimal treatment regimen. To answer these questions, applicants ran a number of studies addressing dosing and timing parameters. The results of one representative study is shown in Figure 5. In this experiment, treatment of animals carrying B16F1 tumors started on day 5 post tumor implantation. The mice received 2 weekly injections of 500 ug/injection for 5 weeks (total of 10 injections and 5 mg per animal) . As can be seen in Figure 5A, TA99 remained effective in inhibiting the growth of tumors when compared to control animals. As can be seen, 2/10 animals in the TA99 group remained tumor free during the course of the study. This is shown graphically in Figure 5B. In addition, only one control mouse remained alive at the end of the study (day 43) compared to 9/10 for the TA99 group. These data indicate that TA99 is effective in lower doses when given over an extended period of time. This suggests the circulating level of TA99 is critical for inhibition of tumor growth and progression.

The results of these studies indicate that TA99 inhibits in vivo tumor growth and progression in the B16/C57B1/6 animal model. This is a highly aggressive tumor in that a subcutaneous injection of 50000 cells is sufficient to induce tumors in 100% of the animals. With TA99, tumor growth and progression are routinely inhibited and, in many cases, a percentage of animals remain tumor free. In addition, tumor inhibition appears to be potentiated by the addition of IL-2 stimulated syngeneic splenocytes which suggest that a cell-based immunological component is involved in the overall inhibitory effect.

Based on the results of these animal studies, applicants propose the following mechanism of action for the inhibitory effect of TA99. the antibody has been shown to be an extremely effective imaging agent in human xenograft models. This probably results from the accretion of gP75 within the tumor mass due to necrosis and concentration of the protein. The in vivo cell depletion studies (CD4+ T cells and NK are required but not CD8+ T cells; unpublished data) and the results of the above animal studies suggest that there is a cellular component that may augment TA99's bioactivity. In applicants' model, TA99 binds to gP75 that has accumulated within the tumor mass. This leads to a high local concentration of antibody within the tumor. The antibody, in turn, binds to and activates infiltration effector cells (e.g., NK, macrophages, granulocytes) through their Fc receptors. These cells are probably the immunological component involved in tumor killing through the release of cytotoxic cytokines such as TNF or by ADCC reactivity. The presence of CD4+ cells may be required to provide additional cytotoxic factors or for the production of IL-2 to promote the expansion of the effector cell populations within the tumor.

Claims

What is claimed is:

1. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of interleukin-2 and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition of claim 1, further comprising stimulated splenocytes from an appropriate host.

3. A pharmaceutical composition of claim 1, further comprising stimulated human peripheral blood mononuclear cells.

4. A pharmaceutical composition of claim 1, further comprising stimulated human lymph node cells.

5. A pharmaceutical composition of claim 2, 3 or 4 wherein the splenocytes are stimulated by interleukin-2.

6. A pharmaceutical composition of claim 1, further comprising lymphokine activated killer cells.

7. A pharmaceutical composition of claim 6, wherein the lymphokine is interleukin-2.

8. A pharmaceutical composition of claim 1, further comprising appropriate natural killer cells which are stimulated.

9. A pharmaceutical composition of claim 2, wherein the natural killer cells are stimulated by interleukin- 2 .

10. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of stimulated splenocytes from an appropriate host and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of stimulated human peripheral blood mononuclear cells and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of stimulated human lymph node cells and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition of claim 10, 11 or 12 wherein the splenocytes are stimulated by interleukin-2.

14. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of lymphokine activated killer cells and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition of claim 14, wherein the lymphokine is interleukin-2.

16. A pharmaceutical composition comprising effective amount of monoclonal antibody TA99, effective amount of appropriate natural killer cells which are stimulated and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition of claim 16, wherein the natural killer cells are stimulated by interleukin-2.

18. A method of treating melanoma in a subject which comprises administering effective amount of the pharmaceutical composition of claim 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16 or 17 to the subject.