Method and compositions for stimulation of an immune response to a differentiation antigen stimulated by an altered differentiation antigen

Tolerance of the immune system for self differentiation antigens can be overcome and an immune response stimulated by administration of a therapeutic differentiation antigen. The therapeutic differentiation antigen is altered with respect to the target differentiation antigen in the individual being treated (i.e., the differentiation antigen to which an immune response is desired) in one of three ways. First, the therapeutic differentiation antigen may be syngeneic with the target differentiation antigen, provided that therapeutic differentiation antigen is expressed in cells of a species different from the individual being treated. For example, a human differentiation antigen expressed in insect or other non-human host cells can be used to stimulate an immune response to the differentiation antigen in a human subject. Second, the therapeutic differentiation antigen may be a mutant form of a syngeneic differentiation antigen, for example a glycosylation mutant. Third, the therapeutic differentiation antigen may be a differentiation antigen (wild-type or mutant) of the same type from a species different from the individual being treated. For example, a mouse differentiation antigen can be used to stimulate an immune response to the corresponding differentiation antigen in a human subject. Administration of altered antigens in accordance with the invention results in an effective immunity against the original antigen expressed by the cancer in the treated individual.

BACKGROUND OF THE INVENTION
 This application relates to a method and compositions for stimulation of an
 immune response to differentiation antigens.
 Differentiation antigens are tissue-specific antigens that are shared by
 autologous and some allogeneic tumors of similar derivation, and on normal
 tissue counterparts at the same stage of differentiation. Differentiation
 antigens have been shown to be expressed by a variety of tumor types,
 including melanoma, leukemia, lymphomas, colorectal, carcinoma, breast
 carcinoma, prostate carcinoma, ovarian carcinoma, pancreas carcinomas, and
 lung cancers. For example, differentiation antigens expressed by melanoma
 cells include Melan-A/MART-1, Pmel 17, tyrosinase, and gp75.
 Differentiation antigen expressed by lymphomas and leukemia include CD19
 and CD20/CD20 B lymphocyte differentiation markers). An example of a
 differentiation antigen expressed by colorectal carcinoma, breast
 carcinoma, pancreas carcinoma, prostate carcinoma, ovarian carcinoma, and
 lung carcinoma is the mucin polypeptide muc-1. A differentiation antigen
 expressed by breast carcinoma is her2/neu. The her2/neu differentiation
 antigen is also expressed by ovarian carcinoma. Differentiation antigens
 expressed by prostate carcinoma include prostate specific antigen,
 prostatic acid phosphatase, and prostate specific membrane antigen.
 Melanocyte differentiation antigens have been shown to be recognized by
 autoantibodies and T cells of persons with melanoma, and to be relevant
 autoantigens. Wang et al., J. Exp. Med. 183: 799-804 (1996); Vijayasaradhi
 et al., J. Exp. Med. 171: 1375-1380 (1990). Unfortunately, in most cases,
 the immune system of the individual is tolerant of these antigens, and
 fails to mount an effective immune response. For the treatment of cancers
 where the tumor expresses differentiation antigens therefore, it would be
 desirable to have a method for stimulating an immune response against the
 differentiation antigen in vivo. It an object of the present invention to
 provide such a method.
 SUMMARY OF THE INVENTION
 It has now been found that the tolerance of the immune system for self
 differentiation antigens can be overcome and an immune response stimulated
 by administration of a therapeutic differentiation antigen. The
 therapeutic differentiation antigen is altered with respect to the target
 differentiation antigen in the individual being treated (i.e., the
 differentiation antigen to which an immune response is desired) in one of
 three ways. First, the therapeutic differentiation antigen may be
 syngeneic with the target differentiation antigen, provided that
 therapeutic differentiation antigen is expressed in cells of a species
 different from the individual being treated. For example, a human
 differentiation antigen expressed in insect or other non-human host cells
 can be used to stimulate an immune response to the differentiation antigen
 in a human subject. Second, the therapeutic differentiation antigen may be
 a mutant form of a syngeneic differentiation antigen, for example a
 glycosylation mutant. Third, the therapeutic differentiation antigen may
 be a differentiation antigen (wild-type or mutant) of the same type from a
 species different from the individual being treated. For example, a mouse
 differentiation antigen can be used to stimulate an immune response to the
 corresponding differentiation antigen in a human subject. Administration
 of altered antigens in accordance with the invention results in an
 effective immunity against the original antigen expressed by the cancer in
 the treated individual.
 A further aspect of the invention are certain compositions and cell lines
 which are useful in practicing the method of the invention. In particular,
 the invention includes non-human cell lines, for example insect cell
 lines, expressing a human differentiation antigen and expression vectors
 useful in generating such cell lines.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides a method for stimulating an immune response
 to a tissue expressing a target differentiation antigen in a subject
 individual. The subject individual is preferably human, although the
 invention can be applied in veterinary applications to animal species,
 preferably mammalian or avian species, as well.
 As used in the specification and claims of this application, the term
 "immune response" encompasses both cellular and humoral immune responses.
 Preferably, the immune response is sufficient to provide immunoprotection
 against growth of tumors expressing the target differentiation antigen.
 The term "stimulate" refers to the initial stimulation of a new immune
 response or to the enhancement of a pre-existing immune response.
 In accordance with the invention, a subject individual is treated by
 administering a therapeutic differentiation antigen of the same type as
 the target differentiation antigen in an amount effective to stimulate an
 immune response. Thus, for example, if the target differentiation antigen
 is the gp75 antigen found in melanoma cells and melanocytes, the
 therapeutic antigen is also a gp75 antigen. It has been found
 experimentally, however, that administration of syngeneic differentiation
 antigens expressed in cells of the same species as the subject individual
 are not effective for stimulating an immune response (See Examples 1 and
 2). Thus, to be effective in the method of the invention, the therapeutic
 differentiation antigen must be altered relative to the target
 differentiation.
 In a first embodiment of the invention, the therapeutic differentiation
 antigen and the target are both from the same species. The therapeutic
 differentiation antigen is produced by expression in cells of a second
 species different from the first species. In a second embodiment of the
 invention, the therapeutic differentiation antigen is a mutant form of a
 syngeneic differentiation antigen. In a third embodiment of the invention,
 the therapeutic differentiation antigen is a xenogeneic differentiation
 antigen. Each of these embodiments will be discussed in turn below.
 Administration of the therapeutic differentiation antigen can be
 accomplished by several routes. First, the therapeutic differentiation
 antigen may be administered as part of a vaccine composition which may
 include one or more adjuvants such as alum, QS21, TITERMAX or its
 derivatives, incomplete or complete Freund's and related adjuvants, and
 cytokines such as granulocyte-macrophage colony stimulating factor, flt-3
 ligand, interleukin-2, interleukin4 and interleukin- 12 for increasing the
 intensity of the immune response. The vaccine composition may be in the
 form of a therapeutic differentiation antigen in a solution or a
 suspension, or the therapeutic differentiation antigen may be introduced
 in a lipid carrier such as a liposome. Such compositions will generally be
 administered by subcutaneous, intradermal or intramuscular route. Vaccine
 compositions containing expressed therapeutic differentiation antigen are
 administered in amounts which are effective to stimulate an immune
 response to the target differentiation antigen in the subject individual.
 The preferred amount to be administered will depend on the species of the
 target individual and on the specific antigen, but can be determined
 through routine preliminary tests in which increasing doses are given and
 the extent of antibody formation or T cell response is measured by ELISA
 or similar tests. T cell responses may also be measured by cellular immune
 assays, such as cytotoxicity, cytokine release assays and proliferation
 assays.
 The mutant syngeneic or xenogeneic therapeutic differentiation antigen may
 also be introduced in accordance with the invention using a DNA
 immunization technique in which DNA encoding the antigen is introduced
 into the subject such that the antigen is expressed by the subject.
 Syngeneic Antigen Expressed in Cells of Different Species
 In accordance with the present invention, an immune response against a
 target differentiation antigen can be stimulated by the administration of
 syngeneic differentiation antigen expressed in cells of a different
 species. In general, the subject being treated will be a human or other
 mammal. Thus, insect cells are a preferred type of cells for expression of
 the syngeneic differentiation antigen. Suitable insect cells lines
 includes Sf9 cells and Schneider 2 Drosophila cells. The therapeutic
 differentiation antigen could also be expressed in bacteria, yeast or
 mammalian cell lines such as COS or chinese hamster ovary cells. Host
 cells which are evolutionarily remote from the subject being treated, e.g.
 insects, yeast or bacteria for a mammalian subject, may be preferred since
 they are less likely to process the expressed protein in a manner
 identical to the subject.
 To provide for expression of the differentiation antigen in the chosen
 system, DNA encoding the differentiation antigen or a portion thereof
 sufficient to provide an immunologically effective expression product is
 inserted into a suitable expression vector. There are many vector systems
 known which provide for expression of incorporated genetic material in a
 host cell, including baculovirus vectors for use with insect cells,
 bacterial and yeast expression vectors, and plasmid vectors (such as
 psvk3) for use with mammalian cells. The use of these systems is well
 known in the art.
 For treatment of humans with a syngeneic differentiation antigen, cDNA
 encoding the human differentiation antigen to be targeted must be
 available. cDNA is produced by reverse transcription of mRNA, and the
 specific cDNA encoding the target differentiation antigen can be
 identified from a human cDNA library using probes derived from the protein
 sequence of the differentiation antigen. The cDNA sequences of various
 human differentiation antigens have been derived by these methods and are
 known in the art. For example, the sequence of human gp75 (also known as
 Tyrosinase-related Protein-1) is known from Vijayasaradhi, S., Bouchard,
 B., Houghton, A. N., "The Melanoma Antigen Gp75 Is the Human Homologue of
 the Mouse B (Brown) Locus Gene Product",. J. Exp. Med. 171: 1375-1380
 (1990); Bouchard et al., J. Exp. Med. 169: 2029-2042 (1989). Other human
 differentiation antigens with known cDNA sequences are gp 100 (also known
 as tyrosinase-related protein-2) (Kawakami et al, Proc. Nat'l. Acad. Sci.
 (USA) 91: 6458-6462 (1994); Adema et al., J. Biol. Chem. 269: 20126-20133
 (1994), and mart-1/melan-a for malignant melanoma; CD19 and CD20 for
 non-Hodgkin's lymphoma; her-2/neu for breast carcinoma (King et al.,
 Science 229: 874-976 (1985); muc-1 for breast, colorectal, lung and
 pancreatic carcinomas (Spicer et al., J. Biol. Chem. 266: 15099-15109
 (1991)); prostate specific membrane antigen, prostate specific antigen,
 and prostatic acid phosphatase for prostate carcinoma (Israeli et al.,
 Cancer Res. 54: 6344-6347 (1994); Monne et al., Cancer Res. 54: 6344-6437
 (1994); Vihko et al., FEBS Lett. 236: 275-281 (1988)).
 The therapeutic differentiation antigen expressed in cells of a different
 species is administered to the subject individual in an amount effective
 to induce an immune response. The composition administered may be a lysate
 of cells expressing the therapeutic differentiation antigen, or it may be
 a purified or partially purified preparation of the therapeutic
 differentiation antigen.
 Mutant forms of Syngeneic Differentiation Antigen
 In the second embodiment of the invention, a mutant form of a syngeneic
 differentiation antigen of a type expressed by the target tumor is used to
 stimulate an immune response directed against the tumor. For example, if
 the tumor is a human tumor that expresses gp75, then a mutant form of
 human gp75 is used as the therapeutic differentiation antigen.
 It will be appreciated by persons skilled in the art that not all mutations
 will produce an antigen which is useful in the method of the present
 invention. For example, large-scale deletions which eliminate important
 epitopes would not be expected to work and are not considered to be
 therapeutic differentiation antigens as that term is used in the
 specification and claims of this application. Less extensive mutations,
 however, particularly those which alter the tertiary and/or quaternary
 structure of the expressed differentiation antigen are within the scope of
 the present invention.
 A preferred type of mutant form of therapeutic differentiation antigen is a
 glycosylation mutant. On any given membrane protein, there will generally
 be one or multiple glycosylation sites, with each site being of different
 importance in its effect on the transport and degradation of the protein.
 For example, in the case of mouse gp75, there are six N-glycosylation
 sites, one of which strongly effects the resistance to protease digestion
 and two others of which are important for permitting export of the protein
 from the endoplasmic reticulum. Glycosylation-mutants that are altered at
 these sites (Asn 96, Asn 104, Asn 181, Asn 304, Asn 350, Asn 385) have
 been prepared using site-directed mutagenesis. These mutations result in
 the conversion of syngeneic proteins which are normally non-immunogenic
 into immunogenic altered antigens.
 Genetic immunization with a glycosylation mutant syngeneic gp75 where
 asparagine at amino acid position 350 is altered to delete the
 glycosylation site at this position was found to stimulate production of
 autoantibodies against an intracellular, early processed form of gp75.
 These autoantibodies did not recognize mature gp75. We have generated
 these same antibodies by immunizing with cells expressing this altered
 protein, i.e., immunization with the altered protein has the same effect.
 Xenogeneic Differentiation Antigens
 In accordance with the present invention, an immune response against a
 target differentiation antigen can be stimulated by the administration of
 xenogeneic differentiation antigen of the same type. Thus, for example, an
 immune response to tumor that expresses gp75 can be stimulated by
 immunization with gp75 derived from a different species which breaks the
 tolerance to the autoantigen. For treatments of humans, preferred
 xenogeneic antigens will be rodent antigens, but could come from other
 mammals such as dog, cat, cow, or sheep, or from birds, fish, amphibian,
 reptile, insect or other more distantly related species.
 Xenogeneic differentiation antigen may be administered as a purified
 differentiation antigen derived form the source organism. Proteins can be
 purified for this purpose from cell lysates using column chromatography
 procedures. Proteins for this purpose may also be purified from
 recombinant sources, such as bacterial or yeast clones or mammalian or
 insect cell lines expressing the desired product. Nucleic acid sequences
 of various differentiation antigens from various non-human sources are
 known, including mouse tyrosinase (gp75) (Yamamoto et al., Japanese J.
 Genetics 64: 121-135 (1989)); mouse gp100 (Bailin et al., J. Invest.
 Dermatol. 106: 24-27 (1996)); and rat prostate-specific membrane antigen
 (Bzdega et al., J. Neurochem. 69: 2270-2277 (1997).
 Xenogeneic differentiation antigen may also be administered indirectly
 through genetic immunization of the subject with DNA encoding the
 differentiation antigen. cDNA encoding the differentiation antigen is
 combined with a promoter which is effective for expression of the nucleic
 acid polymer in mammalian cells. This can be accomplished by digesting the
 nucleic acid polymer with a restriction endonuclease and cloning into a
 plasmid containing a promoter such as the SV40 promoter, the
 cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) promoter.
 The resulting construct is then used as a vaccine for genetic
 immunization. The nucleic acid polymer could also be cloned into plasmid
 and viral vectors that are known to transduce mammalian cells. These
 vectors include retroviral vectors, adenovirus vectors, vaccinia virus
 vectors, pox virus vectors and adenovirus-associated vectors.
 The nucleic acid constructs containing the promoter, antigen-coding region
 and intracellular sorting region can be administered directly or they can
 be packaged in liposomes or coated onto colloidal gold particles prior to
 administration. Techniques for packaging DNA vaccines into liposomes are
 known in the art, for example from Murray, ed. "Gene Transfer and
 Expression Protocols" Humana Pres, Clifton, N.J. (1991). Similarly,
 techniques for coating naked DNA onto gold particles are taught in Yang,
 "Gene transfer into mammalian somatic cells in vivo", Crit. Rev. Biotech.
 12: 335-356 (1992), and techniques for expression of proteins using viral
 vectors are found in Adolph, K. ed. "Viral Genome Methods" CRC Press,
 Florida (1996).
 For genetic immunization, the vaccine compositions are preferably
 administered intradermally, subcutaneously or intramuscularly by injection
 or by gas driven particle bombardment, and are delivered in an amount
 effective to stimulate an immune response in the host organism. The
 compositions may also be administered ex vivo to blood or bone
 marrow-derived cells (which include APCs) using liposomal transfection,
 particle bombardment or viral infection (including co-cultivation
 techniques). The treated cells are then reintroduced back into the subject
 to be immunized. While it will be understood that the amount of material
 needed will depend on the immunogenicity of each individual construct and
 cannot be predicted a priori, the process of determining the appropriate
 dosage for any given construct is straightforward. Specifically, a series
 of dosages of increasing size, starting at about 0.1 ug is administered
 and the resulting immune response is observed, for example by measuring
 antibody titer using an ELISA assay, detecting CTL response using a
 chromium release assay or detecting TH (helper T cell) response using a
 cytokine release assay.
 The invention will now be further described with reference to the
 following, non-limiting examples.
 EXAMPLE 1
 C57BL/6 mice were immunized with a) syngeneic gp75.sup.+ B16 melanoma cells
 (which express a non-mutant b locus protein); b) syngeneic B16 cells
 expressing IL-2, GM-CSF and IFN-.gamma.; c) syngeneic gp75.sup.- B16
 melanoma variant, B78H.1 and syngeneic fibroblasts transfected with cDNA
 expressing the mouse b allele; d) hydrophilic peptides of gp75 conjugated
 to carrier protein; and e) full length gp75 glycoprotein purified from
 syngeneic melanoma cells. Cells, purified glycoprotein or peptides were
 combined with adjuvants, including Freund's adjuvant, a mixture of
 bacterial cell wall skeletons and an endotoxin derivative (DETOX), and a
 saponin component (QS21). Immunizations were tested by intraperitoneal,
 subcutaneous and intradermal routes. After immunizations, mice were
 assessed for antibodies against gp75 by ELISA, inmmunoprecipitation and
 Western blots, and for cytotoxic T lymphocytes (CTL) to B16 using a
 .sup.51 Cr-release cell-mediated cytotoxicity assay. As summiarized in
 Table 1, no antibodies or CTL against gp75 were detected after any of
 these immunization strategies, supporting the conclusion that C57BL/6
 maintain tolerance to the gp75 glycoprotein.
 EXAMPLE 2
 As shown in Example 1, syngeneic C57BL/6 mice immunized with either
 cell-associated or purified forms of gp75 protein did not produce
 autoantibodies to gp75. We next assessed whether gp75 encoded by cDNA
 delivered into the dermis of syngeneic C57BL/6 mice by particle
 bombardment would induce an autoantibody response.
 C57BL/6 mice were genetically immunized with cDNA encoding full-length
 syngeneic gp75 under the control of a CMV promoter once a week for five
 weeks. Sera from these mice were then assessed for autoantibodies against
 gp75 by immunoprecipitation as described in the Materials and Methods. No
 mouse (0/28) had detectable antibodies, indicating that C57BL/6 mice
 maintained their tolerance to the syngeneic protein.
 EXAMPLE 3
 A baculovirus expression vector encoding full length murine gp75 was
 constructed and isolated in collaboration with Dr. Charles Tackney
 (Imclone, New York, N.Y.) using standard techniques. Summers & Smith, "A
 manual for methods for baculovirus vectors and insect cell culture
 procedures", Texas Agricultural Experiment Station Bulletin No 1555
 (1987); Lucklow & Summers, Biotechnology 6:47-55 (1988). Briefly, the 1.8
 kb EcoRI fragment of pHOMERB2 encoding murine gp75 was subcloned into a
 baculovirus expression vector related to pBbac produced by Stratagene,
 Inc, and the expression vector introduced into baculovirus. Spodoptera
 frugiperda Sf9 insect cells were coinfected with this virus construct and
 wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and
 recombinant AcNPV expressing mouse gp75 was generated by homologous
 recombination. After plaque purification, Sf9 cells were infected with the
 recombinant virus and clones expressing high levels of gp75 were
 identified by screening with an antibody against gp75. These cell lines
 were used for immunization studies.
 C57BL/6 mice were immunized with lysates of insect Sf9 cells expressing
 either syngeneic gp75 in a baculovirus vector (gp75/Sf9) or wild-type
 baculovirus (wt/Sf9). Mice immunized with gp75/Sf9 lysates (1 or
 5.times.10.sup.6 cells) developed autoantibodies to gp75 with (120/120
 mice) or without (25/28 mice) Freund's adjuvant. No antibodies were
 detected after immunization with wt/Sf9 (0 of 46 mice). Autoantibodies
 appeared after two to four immunizations, lasted for more than four months
 after the last immunization, and reacted with gp75 expressed in syngeneic
 melanocytic cells (B16F10 and JBRH melanomas). Antibodies were IgG class,
 based on reactivity with rabbit anti-mouse IgG and protein A, and
 copurification of antibody reactivity with IgG fractions from sera.
 The difference in immunogenicity between gp75/Sf9 and mouse gp75 was not
 due simply to quantitative differences in the amount of gp75 in the two
 preparations. 8.times.10.sup.6 B16 melanoma cells contained 20 .mu.g of
 gp75, compared to only 14 .mu.g in 1.times.10.sup.6 gp75/Sf9 cells. Also,
 10 .mu.g of purified mouse gp75 mixed with wt/Sf9 lysates did not induce
 autoantibodies. Although Sf9 cells can apparently provide an adjuvant
 effect (Prehaud et al., Virology 173: 390-399 (1989); Ghiasi et al., J.
 Gen. Virology 73: 719-722 (1992)), these results show that other
 differences between gp75 produced in mouse cells versus insect cells (for
 instance carbohydrate structures) were necessary to induce autoantibodies.
 EXAMPLE 4
 In contrast to immunization with gp75/Sf9 lysates, immunization with
 purified gp75 (12 .mu.g) produced in gp75.Sf9 insect cells plus Freund's
 adjuvant induced autoantibodies that recognized 68/70 kDa early processed
 forms of gp75. This form of gp75 contained only immature, high mannose
 N-linked carbohydrates, which results in localization of the molecule to
 the endoplasmic reticulum or cis Golgi compartment.
 EXAMPLE 5
 Mice were immunized with the gp75.sup.+ human melanoma cell line SK-MEL-19
 with Freund's adjuvant and evaluated for the development of autoantibodies
 to murine gp75. All of the mice (20/20) developed autoantibodies. There
 was no response without adjuvant (0/5 mice), and no antibodies to gp75
 were detected in sera of 12 mice immunized with gp75.sup.- human melanomas
 SK-MEL-131 or SK-MEL-37 plus Freund's adjuvant. Three of five mice
 immunized with purified human gp75 (10 .mu.g per dose for five
 immunizations) with Freund's adjuvant developed autoantibodies to gp75,
 although the antibody responses were generally weaker, possibly due to the
 lower amount of purified gp75 used compared to the amount of gp75 in
 SK-MEL-19 lysates. Thus, administration of human gp75 broke the tolerance
 to gp75 in C57BL/6 mice.
 EXAMPLE 6
 B16 melanoma cells and normal melanocytes in C57BL/6 mice express GP75, the
 wild-type b allele of the brown locus. As described above, the product of
 this locus is recognized by sera from syngeneic mice immunized with mouse
 gp75 expressed in gp75/Sf9 cells and human gp75. We have previously shown
 that passive transfer of mouse monoclonal antibody against gp75 into mice
 bearing B16F10 tumors leads to tumor rejection. Hara et al., Int. J.
 Cancer 61: 253-260 (1995). To determine whether the autoimmune responses
 observed conferred similar protection against tumors, the in vivo effects
 of immune recognition of gp75 were investigated using a syngeneic tumor
 model.
 Mice (5 mice per group) were injected subcutaneously with gp75/Sf9 lysates
 (5.times.10.sup.6 gp75/Sf9 cells) concurrently with 10.sup.5 B16F10
 melanoma cells administered intravenously and the occurrence of lung
 metastases 14 days after tumor challenge was monitored. Mice immunized
 with wt/Sf9 cells and unimmunized mice were used as controls. The results
 are summarized in FIG. 1. As shown, mice immunized with gp75/Sf9 lysates
 were substantially protected against formation of lung metastases compared
 to the controls. Significant protection (53% decrease in lung metastases)
 was also observed when immunization was carried out 4 days after the tumor
 challenge as metastases become established. There was no significant
 protection in mice immunized with wt/Sf9 lysates compared to the
 unimmunized control.
 Passive transfer of serum from mice immunized with gp75/Sf9 to five
 unimmunized mice produced a 68% decrease in lung metastases compared to
 mice treated with an equivalent amount of normal mouse serum (p=0.02),
 supporting the conclusion that tumor protection was at least partially
 mediated by humoral mechanisms.
 Mice immunized with human gp75.sup.+ SK-MEL-19 were also markedly protected
 against B16F10 melanoma compared to unimmunized mice. (4+/-7 metastases in
 immunized mice versus 275+/-77 lung metastases in control mice--6 mice per
 group). Immunization with gp75.sup.- melanoma SK-MEL-131 did not introduce
 tumor protection against B16F10 melanoma, although recognition of other
 xenogeneic antigens other than gp75 could not be critically assessed.
 Mice immunized against the immature, early processed form of gp75, using
 purified gp75 from gp75/Sf9 cells were not significantly protected against
 B16F10 metastases (366+/-78 metastases in four immunized mice versus
 412+/-94 metastases in five unimmunized control mice). However one mouse
 in this group did eventually develop autoantibodies against mature gp75
 and was protected against lung metastases (only 21 metastases).
 EXAMPLE 7
 C57BL/6 mice were genetically immunized with cDNA encoding full length
 human gp75 under control of the control of a CMV promoter once a week for
 five weeks by gene gun injection. As controls, mice were injected with
 full length syngeneic mouse gp75 under the control of the CMV promoter,
 with a glycosylation mutant of gp75 (gly31) or null DNA. Four weeks after
 the final immunization, the mice were injected through the tail vein with
 2.times.10.sup.5 B16F10LM3 melanoma cells. One group of treated mice were
 also challenged with melanoma cells. Twenty-one days after tumor
 challenge, mice were sacrificed and surface metastatic lung nodules were
 scored. There were ten mice in the untreated group, 9 mice in each of the
 null and mouse gp75 groups, 8 mice in the gly31 group and 19 mice in the
 human gp75 group. The importance of CD4, CD8 and NK cells was also tested
 by depletion of using monoclonal antibodies (rat mAb GK1.5 for CD4; mAb
 53.6.7 for CD8 and mAb PK1.36 for NK1.1). The necessity of CD4 T cells was
 also assessed by looking for tumor rejection in CD4 knock-out mice after
 in vivo transfer of the human gp75 gene by gene gun.
 As shown in FIG. 2, mice immunized with xenogeneic human gp75 were found to
 be significantly protected from lung metastases (mean 41.+-.15 metastases)
 when challenged with B16F10LM3 melanoma (p&lt;0.0001), with an 84%
 decrease in lung nodules as compared with control mice. Syngeneic mice
 that received in vivo gene transfer of the glycosylation mutant mouse gp75
 were not significantly protected from B16F10LM3 tumor challenge (mean
 300.+-.12 metastases), nor were those that were delivered control DNA
 (mean 292.+-.15 metastases) by particle bombardment or were left untreated
 (mean 307.+-.20 metastases) (p&gt;0.45). CD8 deletion did not alter tumor
 rejection, although depletion of CD4.sup.+ (by mAb or knock-out) and
 NK1.1.sup.+ cells did result in a reduction in level of protection
 achieved. Thus these latter cells may play a role in the protection
 against tumors achieved using genetic immunization with xenogeneic DNA.