WO1996040249A1

WO1996040249A1 - Therapeutic uses of ta99

Info

Publication number: WO1996040249A1
Application number: PCT/US1996/010038
Authority: WO
Inventors: Alan N. Houghton
Original assignee: Memorial Sloan Kettering Cancer Center
Current assignee: Memorial Sloan Kettering Cancer Center
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19
Anticipated expiration: 1997-12-07
Also published as: AU6171196A

Abstract

The invention provides a pharmaceutical composition comprising effective amount of TA99 monoclonal antibody and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising effective amount of a humanized TA99 antibody and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising effective amount of an antibody capable of competitively inhibiting the binding TA99 to gp75 and a pharmaceutically acceptable carrier. This invention further provides a method of treating tumor comprising administering effective amount of the above-described pharmaceutical compositions. In an embodiment, the tumor is a melanoma.

Description

THERAPEUTIC USES OF TA99

This invention disclosed herein was made with United States Government support under National Institute of Health Grants, R01 CA56821 and ROl CA33049. Accordingly, the United States Government has certain rights in this invention.

Background of the Invention

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Experimental evidence shows that the immune system can recognize antigens expressed by cancer cells. Recently the isolation of genes encoding cancer antigens has led to the identification of these molecules [1-3] . From these studies, an emerging paradigm is that the immune repertoire recognizes a set of self antigens, termed differentiation antigens, expressed by malignant cells but also by their normal cell counterparts [3-5] .

The immune response to melanoma has been studied more extensively than to any other human cancer. Dissection of the immune response to melanoma has shown that differentiation antigens expressed by normal melanocytes and related neuroectoderm-derived cells are dominant antigens recognized by the immune system [3,5-16] . Among the best characterized melanoma autoantigens are proteins of the tyrosinase family. Tyrosinase (the product of the albino locus) is the prototype molecule, and other members include gp75 or tyrosinase-related protein-1 (the product of the Jrown locus) and gplOO (the presumed product of the silver locus) [17-19] . These molecules, plus the MelanA/MART-1 antigen, are all melanocyte- specific molecules recognized by the immune system of melanoma patients [8,9,11-16] . An important question is whether immune recognition of self (differentiation antigens) can lead to tumor rejection. In particular, can immunity to melanocytes actually lead to rejection of melanoma and are there sequelae in normal tissues that contain melanocytes.

The immune system can recognize differentiation antigens that are selectively expressed on malignant cells and their normal cell counterparts. However, it is uncertain whether immunity to differentiation antigens can effectively lead to tumor rejection. The mouse brown locus protein, gp75 or tyrosinase-related protein-1, is a melanocyte differentiation antigen expressed by melanomas and normal melanocytes. The gp75 antigen is recognized by autoantibodies and autoreactive T cells in persons with melanoma. To model autoimmunity against a melanocyte differentiation antigen, mouse antibodies against gp75 were passively transferred into tumor- bearing mice. Passive immunization with a mouse monoclonal antibody against gp75 induced protection and rejection of both subcutaneous tumors and lung metastases in syngeneic C57BL/6 mice, including established tumors. Passive immunity produced coat color alterations but only in regenerating hairs. This system provides a model for autoimmune vitiligo and shows that immune responses to melanocyte differentiation antigens can influence mouse coat color. Immune recognition of a melanocyte differentiation antigen can reject tumors, providing a basis for targeting tissue autoantigens expressed on cancer.

Brief Description of the Figures

Figure 1 Protective immunity induced by mAb TA99 against B16F10 melanoma cells. Melanoma cells (5 x 10⁴) were injected subcutaneously in 0.1 ml normal saline into the flank of C57BL/6 mice. Mice were treated intraperitoneally with (A) control mouse IgG2a mAb UPC10 or (B) mAb TA99, injected at a dose of 150 μg diluted in

0.3-0.4 ml of normal saline three times per week (days 0, 2, 4, 7, 9 and 11) . TA99 mAb was purified from ascites by protein A affinity column. There were eight mice in each group. Tumors were assessed three time per week by palpation and inspection. Tumor size (ordinate) was measured in millimeters using calipers. The longest surface diameter (a) and its perpendicular width (b) were measured, and tumor size reported as a x b. Time (abscissa) is presented in days from tumor challenge

(day 0) . Mice were monitored at least four times per week. Treatment with mAb TA99 produced a significant increase

(p<0.0001, Bonferroni two sided t test) in proportion of tumor-free mice. This experiment was repeated three time. In separate experiments, injection with control mAb UPC10 did not induce any difference in growth of B16F10 melanoma compared to untreated control mice. All animal experiments and care was in accordance with institutional guidelines. Each symbol in (A) represents an individual mouse. In group (B) , 8 mice were treated with mAb TA99, and only one mouse developed palpable tumor (1) .

Figure 2 Protective immunity by different doses of mAb TA99 against B16F10 melanoma. B16F10 cells were injected subcutaneously in the flank of C57BL/6 mice (see legend of Figure 1) . Mice were treated intraperitoneally with control mAb UPC10 150 μg (1) or mAb TA99 at doses of 150 μg (n) , 75 μg (A) or 37.5 μg (V) per dose as described in the legend of Figure 1. There were six to eight mice in each group. Tumors were assessed four times per week by palpation and inspection. Mice were scored as tumor-free or tumor- bearing and the proportion of tumor- bearing mice (ratio of tumor-bearing/total number of mice per group) was calculated for each time point.

Figure 3 Anti-tumor effects of mAb TA99 on B16F10 lung metastases. Syngeneic C57BL/6 mice were injected intravenously through the tail vein with lxlO⁵ B16F10 melanoma cells in 0.1 ml sterile saline. Mice were treated intraperitoneally with control mAb UPC10 or mAb TA99, 150 μg dose, three times per week (days 0, 2, 4, 7, 9 and 11) . At 18 days after tumor challenge, mice were sacrificed and surface lung metastases were scored and counted as black nodules under a dissecting microscope. There were eight mice in each group. This experiment was repeated five times. Error bars represent one standard deviation.

Figure 4 Anti-tumor effects of mAb TA99 on established B16F10 lung metastases. C57BL/6 mice were injected intravenously with B16F10 melanoma (see legend of Figure 3) . Mice (five to nine mice in each group) were treated intraperitoneally with either control mAb UPC10 or mAb TA99 600 μg/dose three times per week for two weeks starting day 0 (6 hours after tumor challenge), day 2, day 4 or day 7. Surface lung metastases were detected by day 4-6 under a dissecting microscope and by day 7-8 by eye. At 18 days after tumor challenge, mice were sacrificed and lung metastases were counted. This experiment was performed three times (daily doses of mAb TA99 between 300 and 1000 μg) . Error bars represent one standard division.

Figure 5 Evaluation of potential effector functions in rejection of B16F10 ling metastases induced by mAb TA99. C57BL/6 mice were challenged intravenously with melanoma cells (see legend of Figure 3) and treated with control mAb UPC10 or mAb TAOO 150 μg dose three times per week (days 0, 2, 4, 7, 9 and 11) for two weeks. Mice were sacrificed on day 18. There were 5 to 7 mice per group. This experiment was repeated twice. Depletion of CD4+ and CD8+ T cell subsets in vivo was accomplished by intraperitoneal administration of rat mAb GK1.5 (anti-CD4) and mAb 2.43 (anti-CD8) (from the American Type Culture Collection, ATCC) . Natural killer cell depletion was performed using mAb PK1.36 (anti-NK-1.1) (from ATCC) . Monoclonal antibody preparations (0.2 ml) were injected intraperitoneally at day -3 and every 7 days thereafter. Throughout experiments, these treatments were shown to deplete respective T cell subpopulation > 98% as determined by indirect immunofluorescence staining and cytofluorometric analysis of peripheral blood, thymus, and spleen. Depletion of

NK cells from splenocytes was demonstrated in 4 hr ⁵¹Cr-release assays with YAC cells as targets (effector-to-target ratios of 100:1, 50:1 and 25:1) . PK1.36 treatment was shown to abrogate Hara et al . NK activity completely. For depletion of complement, mice were injected intraperitoneally with 10 U cobra venom factor (Diamedex, Miami, FL) on day -1 and every 4 days. Depletion of complement was verified using sensitized rabbit RBC (17) . Error bars represent one standard deviation.

Figure 6 Alteration of coat color in C57BL/6 mice treated with mAb TA99. Mice were (6-8 weeks old) were depilated on day -1 over the posterior flank. The same results were observed if the coat was plucked manually or depilated with hair remover (JMC, Inc., Tokyo) . On day 0, mice started treatment with either mAb TA99 (left mouse) or mAb UPC10 (right mouse) three times per week for two weeks (see 5 schedule in Figure 1) at individual daily doses of 600 μg. The line demarcating the upper border of the shaved coat (dorsal trunk of the animal) can be seen in the control animal (right mouse) . The coat of

10 mice treated with mAb TA99 showed patchy depigmentation in areas of regenerating hairs but not in the non-depilated coat, while no alteration in coat color was observed in mice treated with isotype

15 control mAb UPC10.

Summary of the Invention

This invention provides a pharmaceutical composition comprising effective amount of TA99 monoclonal antibody and a pharmaceutically acceptable carrier.

This invention also provides a pharmaceutical composition comprising effective amount of a humanized TA99 antibody and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising effective amount of an antibody capable of competitively inhibiting the binding TA99 to gp75 and a pharmaceutically acceptable carrier.

This invention further provides a method of treating tumor comprising administering effective amount of the above-described pharmaceutical compositions. In an embodiment, the tumor is a melanoma.

Detailed Description of the Invention

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 sμch 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 which express gp75. 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 a humanized TA99 antibody and a pharmaceutically acceptable carrier. Methods to synthesize a humanized antibody from a mouse monoclonal antibody are well known in the art. The complete backbone of the mouse antibody may be changed to human backbone.

This invention also provides a pharmaceutical composition comprising effective amount of an antibody capable of competitively inhibiting the binding TA99 to gp75 and a pharmaceutically acceptable carrier. In an embodiment, this antibody is a humanized antibody.

This invention provides a pharmaceutical composition comprising an effective amount of an antibody capable of binding the epitope recognized by TA99 antibody and a pharmaceutically acceptable carrier.

"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

Experimental Details 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

Mice and Tumors. C57BL/6 (6-8 week old females) were obtained from the Jackson Laboratory (Bar Harbor, ME) . B16F10 is a mouse melanoma cell line of C57BL/6 origin kindly provided by Dr. Isaiah Fidler (MD Anderson Cancer Center, Houston, TX) [20] . B78H.1 is a variant of B16 melanoma that does not express the gp75 antigen. JBRH is a melanoma from C57BL/6 provided by Dr. P. Livingston

(Memorial Sloan-Kettering, NY) . Other tumors derived from C57BL/6 mice include the RMA and EL-4 lymphomas,

Lewis lung carcinoma, and MC58 sarcoma (from Dr. N. Nicolic-Zugic, Memorial Sloan-Kettering, NY) . All animal experiments and care was in accordance with institutional guidelines.

For subcutaneous tumors, tumor cell were injected subcutaneously in the mouse flank. B16F10 melanoma cells

(5 x 10⁴ cells), B78H.1, RMA, EL-4, JBRH and Lewis lung carcinoma (10⁴-10⁶ cells) were injected subcutaneously in 0.1 ml normal saline into the flank of syngeneic C57BL/6 mice. Tumors were checked at least four times per week by palpation and inspection. For each tumor type, palpable tumors formed in 10-21 days. For B16F10 melanoma lung metastases, C57BL/6 mice were injected intravenously through the tail vein with 1x10^s B16F10 melanoma cells in sterile saline. For depilation experiments, C57BL/6 mice were depilated on the day before starting treatment over the posterior flank and observed for coat color. The same results were observed if the coat was plucked manually under anesthesia or depilated with hair remover (JMC, Inc. , Tokyo) . Mice were treated intraperitoneally with mAb TA99 or control mouse IgG2a mAb UPC10 diluted in 0.3-0.4 ml of normal saline. TA99 mAb was purified from mouse ascites by protein A affinity column. F(ab')₂ fragments of mAb TA99 were produced by digestion with pepsin and purification over protein A Sepharose. Control IgG2a mAb UPC10 was from Sigma Chemical Co. (St. Louis, MO) . Injection with control mAb UPC10 did not induce any difference in growth of B16F10 melanoma compared to untreated control mice. For subcutaneous tumor measurements, the longest surface length (a) and its perpendicular width (b) were measured, and tumor size reported as a x b. For lung metastases, at 16-20 days after tumor challenge mice were sacrificed and surface lung metastases were scored and counted as black nodules under a dissecting microscope. Surface lung metastases were detected by day 4-7 under a dissecting microscope and by day 7-10 by eye. For histologic evaluation, tissues and tumors were fixed in formalin solution, blocked in paraffin, sectioned every 4 microns and stained with hematoxylin and eosin. Statistical analysis of tumor growth was performed using the Student's ϋ test or Bonferroni two sided t test, a conservative analysis to allow for multiple comparisons.

Antibody treatments in vivo . Depletion of T cells in vivo was accomplished by intraperitoneal administration of rat mAb GK1.5 (anti-CD4; IgG2b) and mAb 2.43 (anti- CD8; IgG2b) , and Thyl.2⁺ cells by mAb 30-H12 [produced by hybridomas from the American Type Culture Collection (ATCC) , Rockville, MD] . These mAb were used as ascites fluids (titer >1:10,000 by staining of mouse thymocytes by flow cytometry) . Monoclonal antibody preparations (0.2 ml) were injected intraperitoneally at day -3 and every 7 days thereafter. Throughout experiments, these treatments depleted respective T cell subpopulations and Thy-1⁺ populations >97% as determined by indirect immunofluorescence staining and cytofluorometric analysis of lymph nodes and thymocytes with mAbs GK1.5 (CD4) , 2.43 (CD8) or 30-H12 (Thyl.2) . Natural killer (NK) cell depletion was performed using mAb PK136 (anti-NK-1.1)

(from ATCC) . Antibody (0.2 ml) was injected intraperitoneally at day -3 and every 7 days thereafter. Depletion of NK cells was assessed by 4 hr ⁵¹Cr-release assays with 5,000 YAC cells as targets and spleen cells as effector cells at effector-to-target ratios of 100:1, 50:1 and 25:1, and depletion was shown to abrogate completely detectable NK activity. For depletion of complement, mice were injected intraperitoneally with 10 U cobra venom factor (Diamedexz, Miami, FL) on day -1 and every 4 days. Depletion of complement in sera of treated mice was verified using lysis of sensitized rabbit RBC

[21] .

Expression of gp75 antigen on B16F10 melanoma.

Intracellular expression of gp75 was determined using mAb TA99 in indirect immunofluorescence assays against B16F10 melanoma cells fixed and permeabilized in methanol :acetone (1:1) at 4° C for 15 min. Intracellular expression was confirmed by immunoelectronmicroscopy using protein A labelled colloidal gold particles as described [22] . Cell surface expression was shown by binding of mAb TA99 to intact, live B16F10 cells by enzyme-linked immunoassay (titer >l/10,000) and by protein A mixed hemadsorption assay (titer >l/5000) [5,22] . Experimental Results and Discussion

Syngeneic mouse tumor models were established to examine whether immunity against antigens expressed on normal melanocytes and melanoma can lead to tumor rejection. The brown locus product, gp75, was the target antigen in these models. The gp75 autoantigen is relevant because it is potentially immunogenic in persons with melanoma, recognized on melanomas by both autoantibodies and autoreactive T cells [8,9] . The mouse mAb TA99 binds to both human and mouse gp75, and reacts with normal melanocytes and melanoma but does not react with other tissues [22] . TA99 mAb has been shown to localize efficiently to melanoma xenografts in mice (tumor:normal tissue ratios >100:1 to 10⁵:1) , showing that systemic administration of antibody against gp75 can specifically localize to tumor sites [23] .

The gp75⁺ B16F10 melanoma is a spontaneously arising tumor that is very weakly immunogenic in syngeneic

C57BL/6 mice. B16F10 cells from fresh tumors express gp75 in melanosomes within the cell and on the cell surface (see Materials and Methods) . Incubation of mAb TA99 (up to 600 mg/ml for seven days) with B16F10 melanoma cells in vi tro did not affect the cell growth, morphology or pigmentation. Mice were challenged subcutaneously with B16F10 melanoma and treated with either mAb TA99 or isotype-matched control mAb UPC10. With this tumor challenge, B16F10 uniformly forms palpable tumors around two weeks. Tumors were rejected in mice treated with mAb TA99 but not in control mice

(Figure 1) . Protection against tumor growth was observed with doses of mAb TA99 as low as 37.5 mg, but optimal protection was seen with doses of 150 mg mAb TA99 or greater (Figure 2) . Tumor protection was observed beyond 50 - 70 days .

This anti-tumor effect was specific for tumors that expressed gp75 antigen. Tumor protection was seen for the gp75⁺ JBRH melanoma after subcutaneous challenge in syngeneic C57BL/6 mice (time to median appearance of tumors delayed by >31 days) . However, no antitumor effects were observed in syngeneic C57BL/6 mice with a gp75^" variant of the parental B16 melanoma (B78H.1 melanoma) , nor with other subcutaneous gp75^" tumors, including EL4 lymphoma, RMA lymphoma, Lewis lung carcinoma, or MC58 sarcoma.

Intravenous injection of B16F10 leads reproducibly to lung metastases [20,24] . Treatment with mAb TA99 markedly reduced the number of lung surface metastases even at individual doses as low as 50 mg (Figure 3A and data not shown) . Injection of F(ab')₂ fragments of mAb TA99 did not have any effect on the number of B16F10 metastases, supporting a crucial role for the Fc portion of mAb TA99 in anti-tumor effects (data not shown) .

Applicants asked whether treatment with mAb TA99 would have antitumor effects in established metastases. Delaying treatment with mAb TA99 for two to seven days after challenge with B16F10 still induced substantial protection against metastases but required higher doses of mAb TA99 (p<0.001, paired Student's t test at seven days) (Figure 3B) . Effects of mAb TA99 were also observed on growth of subcutaneous B16F10 melanoma when treatment was delayed four days after tumor challenge (median time to appearance of tumor increased by 14 days) , and slightly but reproducibly when treatment was delayed seven days (median time to appearance of tumor increased six days) .

Histologic examination of residual B16F10 subcutaneous lesions and metastatic lung lesions in mice treated with mAb TA99 showed occasional infiltration of lymphocytes and macrophages, compared to no mononuclear or inflammatory infiltrates observed in control mice. To assess what components of the immune or inflammatory system might be involved in tumor rejection, T lymphocyte subsets, complement, or natural killer (NK) cell populations were depleted before challenge with tumor cells. Depletion of CD8⁺ T cells and complement did not alter tumor rejection mediated by mAb TA99 (Figure 4 shows results for lung metastases; data not shown for subcutaneous tumors) . Depletion of CD4⁺ cells partially decreased rejection of B16F10 lung metastases by mAb TA99 (Figure 4) (depletion of CD4⁺ cells without mAb TA99 treatment did not affect the number of B16F10 metastases; data not shown) , but did not affect growth of subcutaneous B16F10 tumors.

Depletion of an NKl.1⁺ cell population appeared to abrogate the protective effect of mAb TA99 in both metastatic B16F10 in the lung (Figure 4) and subcutaneous B16F10 tumors (data not shown) , supporting a role for NK cells in tumor rejection mediated by mAb TA99. NKl .1+ cells appear to provide natural immunity against B16F10 lung metastases [24] . Applicants further examined the role of NKl.l" cells in the lung metastases model (using the same experimental design described in Figure 5 with 6-8 mice/group) : 1) Depletion of NKl .1+ cells from C57BL/6 mice led to a significant increase in the number of metastases (mean 314.+58 metastases) compared to control undepleted mice (136.+25 metastases) ; 2) Treatment with mAb TA99 markedly decreased metastases (12±11 metastases) ; and 3) The number of lung metastases in mice treated with mAb TA99 and also depleted of NKl .1⁺ cells was significantly greater (mean 186±42) than mice treated with TA99 alone (12±11) , but not as great as the number of metastases in untreated NKl.1-depleted mice (314±58) .

These results suggested that other components of the host in addition to NK cells, such as CD4+ cells (Figure 4) , participated in rejection of lung metastases mediated by mAb TA99. It is possible that NKl.1⁺ cells binding mAb TA99 through Fc receptors secondarily activated CD4^* cells to participate in tumor rejection. Alternatively, CD4⁺ T cells might be activated by antigen-presenting cells through internalization and presentation of antigen- antibody complexes.

Mice treated with mAb TA99 were examined for changes in pigmentation and other autoimmune-type manifestations. The coat of animals remained black except if animals were depilated to prepare skin sites for tumor injection. Pigmented melanocytes on the trunk of adult mice are found in hair bulbs. Regenerating hairs on the trunk were depigmented in 13 of 13 tumor-bearing mice treated with mAb TA99 at 300-900 mg per dose (three times per week for two weeks) but not in mice treated with control mAb UPC10

(0 of 36 mice) or untreated control mice (0 of 30 mice) .

Depigmentation was not related to injection of tumor cells; treatment of depilated C57BL/6 mice without injection of tumor cells still led to depigmentation in 12 of 12 mice treated with 300-1000 mg dose of mAb TA99

(Figure 5) . At TA99 doses <150 mg, depigmentation was not observed (0 of 54 mice) . Thus, the threshold dose of mAb TA99 required for coat color changes was five-fold greater than the threshold required for antitumor effects in tumor protection experiments. Histologic sections of skin from mice treated with mAb TA99 showed depigmented hair follicles and regenerating hairs in previously depilated areas. The bulbs of white hairs did not contain pigment, and follicles lacked pigmented melanocytes. There were no signs of decrease in pigmentation or pigmented granules, inflammation, or changes in cellular morphologies or tissue architecture in the eyes (choroid and retina) of mice treated with mAb TA99. There were no detectable alterations in behavior or weights of mice during or after treatment. In vivo depletion with a mAb against Thyl.2 showed that Thyl⁺ cells were necessary for depigmentation, but NK1.1⁺ cells, complement, and the individual CD4⁺ or CD8⁺ T cell subsets were not necessary.

These findings suggest that autoimmunity directed against tyrosinase-related proteins and other antigens expressed by melanocytes can influence coat color in mice. This implies that coat color can be regulated at the level of a host response in addition to previously defined genetic controls at the cellular level. The induction of concomitant tumor rejection and autoimmunity recalls a relevant clinical observation in persons with metastatic melanoma who develop vitiligo. The spontaneous appearance of vitiligo has been associated with an improved prognosis in persons with metastatic melanoma

[25,26] . A related observation is that the immune system of patients with vitiligo can recognize tyrosinase and other specific antigens expressed by melanocytes [27, 28] . This mouse model has recapitulated an association between melanoma and vitiligo, showing that a biologic response to the tyrosinase family of proteins in melanocytes of the skin can mediate melanoma rejection.

In summary, an increasing body of data suggests that immune recognition of melanoma in humans is directed most frequently against molecules expressed by melanocytes or other neural crest-derived cell types (it should be pointed out that this perceived "immune repertoire" against melanoma can be biased by the use of selected in vi tro assays and may not be a random sampling) . These experiments suggest that the threshold for depigmentation, a potential autoimmune manifestation, can be greater than for tumor protection and that the biologic state of melanocytes may be important in the development of autoimmune signs. Passive transfer of mAb against gp75 was able to lead to rejection even of established B16F10 tumors in the lung (similar effects have been observed with mAb treatment against other antigens expressed by B16F10) [29] . Applicants have shown that passive immunity against melanocytes can lead to effective rejection of even established tumors. Melanoma is not the only tumor type where differentiation antigens are recognized by the immune system. Immune recognition of differentiation antigens has also been detected in patients with breast, colon, and pancreas carcinomas [30- 32] .

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Claims

What is claimed is :

1. A pharmaceutical composition comprising an effective amount of TA99 monoclonal antibody and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition comprising an effective amount of a humanized TA99 antibody and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising an effective amount of an antibody capable of competitively inhibiting the binding of TA99 antibody to gp75 and a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising an effective amount of an antibody capable of binding the epitope recognized by TA99 antibody and a pharmaceutically acceptable carrier.

A method of treating tumor in a subject comprising administering to the subject the pharmaceutical composition of claim 1, 2, 3 or 4.

6. The method of claim 5, wherein the tumor is a melanoma.