# Patent

## Title
Her2 DNA vaccine as adjunct treatment for cancers in companion animals

## Abstract

The application discloses therapeutic vaccines based upon the “pING” DNA plasmid vector expressing the gene encoding the rat Her2 protein. Vaccines according to the instant disclosure are used as an adjunct treatment for surgery, radiation and/or chemotherapy for dogs and cats with cancers that over express the Her2 antigen, and prolong the post-surgical disease free interval and/or survival time. Also included are therapeutically effective methods of immunization using said vaccines.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of, and claims priority to, U.S. application Ser. No. 13/272,651, filed on Oct. 13, 2011, which claims benefit of U.S. provisional application Ser. No. 61/394,505, filed Oct. 19, 2010.


BACKGROUND OF THE INVENTION
This application relates to compositions for treatment of differentiation antigen-dependent cancers and to methods of using such compositions. The invention utilizes compositions containing xenogeneic differentiation antigens, which are associated with cancers to provide effective therapy.
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, Pmel17, 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, for example, breast carcinoma is Her2 (synonyms: Her2/neu, ECBB2, ErbB2, c-erb-2), which is a gene coding for a tyrosine kinase receptor that is a member of the family of epidermal growth factor receptors (De Maria et al., 2005). Over expression of Her2 has been demonstrated in mammary gland tumors of both the cat (Winston et al., 2005) and the dog (Rungsipipat et al., 2008). Winston et al. (2005) used existing assay methods (HERCEPTESTTM, Dako USA, Carpinteria, Calif.; NCL-CB11, Novocastra, Newcastle, UK) to successfully grade levels of Her2 expression on feline mammary tumors as 0=minimal/absent, 1=weak, 2=moderate, or 3=intense. The HERCEPTESTTM and NCL-CB11 assays identified 27 and 23 cats respectively, out of 30 examined, as having grade 2 or 3 Her2 expression in mammary tumor samples.
In addition to successfully grading levels of Her2 over expression in feline mammary tumors, Winston et al. (2005) used the HERCEPTESTTM to detect low levels of Her2 expression in normal feline epithelial tissues and cell types including: hair follicle, mammary gland, gastric pit, salivary gland duct, renal cortical and medullary tubules, colonic and small intestinal crypt, brain, pancreatic duct and islets, splenic macrophages, adrenal cortex, hepatocytes, and testicular Leydig's cells. Expression of Her2 has been documented on a range of human epithelial cell types including gastro-intestinal, respiratory, reproductive, urinary, skin, mammary and placenta (Press et al., 1990). These findings indicate that the expression of Her2 is common in a range of tissue types of humans and cats. The finding of Her2 over expression in dog mammary tumors suggests this species would share expression characteristics identified in humans and cats. Existing assays and reagents can serve as tools to screen expression levels of Her2 in companion animal cancers in order to justify treatment with the Her2 cancer vaccine.
Unfortunately, in most cases, the immune system of the individual is tolerant of such differentiation antigens, and fails to mount an effective immune response. Several technologies have been considered to address this challenge: (cytokines as genetic adjuvants (Chang et al., 2004), xenogeneic vaccination (Pupa et al., 2005), electrotransfer (Quaglino et al., 2004), combination with chemotherapy (Bernhardt et al., 2002). Although results were encouraging, greater efficacy was required for these approaches to be considered a key component of a first-line therapeutic strategy. Further, recent findings indicate both antibody and cell-mediated immunity are required for tumor eradication post immunization, perhaps explaining, in part, the lack of success in the field (Orlandi et al., 2007). Therefore, for the treatment of cancers where the tumor expresses differentiation antigens therefore, it would be desirable to have a method for stimulating a therapeutically effective immune response against the differentiation antigen in vivo. It an object of the present invention to provide such a method.
REFERENCES


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All of the above-mentioned applications, patents and references are herein incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
It has now been found that the tolerance of the immune system for self-derived target differentiation antigens can be overcome and an immune response stimulated by administration of a xenogeneic differentiation antigen (wild-type or mutant) of the same type from a species different from the subject being treated (U.S. Pat. No. 6,328,969 & U.S. Pat. No. 7,556,805, to Sloan-Kettering, both incorporated by reference herein). For example, a rat differentiation antigen can be used to stimulate an immune response to the corresponding differentiation antigen in a canine 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 subject. Thus, in accordance with a first aspect of the invention, there is provided a method for treating in a mammalian subject, comprising the step of administering to the subject an immunologically-effective amount of a xenogeneic mammary gland tumor-associated differentiation antigen.
Therapeutic differentiation antigens based on mammary gland carcinoma/tumor-associated differentiation antigens are used in accordance with the invention to treat, for example, mammary gland carcinoma post-surgical removal of tumors in subjects suffering from said cancers. In one embodiment of the invention, a plasmid comprising a sequence encoding a xenogeneic tyrosine kinase receptor, for example rat tyrosine kinase receptor, under the control of a suitable promoter, is administered to a subject. For example, dogs have been treated using plasmids comprising a DNA sequence encoding rat tyrosine kinase receptor with pronounced clinical benefit.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows overall survival time post-immunization and surgical resection of MGT;
FIG. 1B shows disease-free survival time post-immunization and surgical resection of MGT;
FIG. 1C shows metastasis-free survival time post-immunization and surgical resection of MGT;
FIG. 2 shows a map of the pcDNA3.1 (+/−) plasmid
FIG. 3 shows a sequence for the pINGhumanTyrosinase plasmid, where the coding sequence for the human tyrosinase has been removed. This is where the rat Her2/neu (nucleotides 17-3799 of SEQ ID NO:1) was inserted to produce rHer2/neu-pING of the instant invention;
FIG. 4 shows a map of the pING plasmid.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for treating mammary gland tumors in a subject by stimulating an immune response to a mammary gland-associated differentiation antigen. The subject is preferably canine or feline, although the invention can be applied to other 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 is treated by administering a xenogeneic differentiation antigen of the same type as a target differentiation antigen expressed by mammary gland tumor cells of the subject in an amount effective to stimulate an immune response. Thus, for example, if the target differentiation antigen is the Her2/neu antigen found in mammary cells, the therapeutic antigen is a xenogeneic Her2/neu antigen.
In one embodiment, the inventive method may include the following steps: (1) immunization to an animal in need of a xenogeneic antigen, for example, the rat Her2/neu as set forth in SEQ ID NO:2 and encoded by nucleotides 106-3885 of the sequence as set forth in SEQ ID NO:1, (2) needle-free priming of immune responses, (3) electrotransfer-based booster, and (4) vaccination after tumor debulking by surgical primary therapy.
In another embodiment, the inventive method is carried out on subjects, including companion animals, without metastasis (i.e. in relatively early stages of mammary carcinoma disease progression).
In some embodiments, the boost comprises administering plasmids encoding xenogeneic antigens, for example those encoding rat Her2 protein (SEQ ID NO:2).
In some embodiments, the xenogeneic antigen is encoded by a nucleotide having favorable nucleotide substitutions with respect to the sequence as set forth in SEQ ID NO:1. Favorable substitutions include any changes that result in improved immune response against the Her2/neu expressed by the cells of the mammary tumor/carcinoma. Substitutions can include existing sequences, such as murine Her2 (SEQ ID NO:3), human Her2 (SEQ ID NO:4), or any other xenogeneic Her2 sequence, or fragment thereof, capable of eliciting a therapeutically effective immune response in a target animal against a Her2-associated mammary carcinoma.
In some embodiments, the boost comprises administering a xenogeneic differentiation antigen.
In other embodiments, the boost comprises administering a syngeneic differentiation antigen.
Xenogeneic differentiation antigen may be administered as a purified differentiation antigen derived from 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.
Administration of the xenogeneic differentiation antigen can be accomplished by several routes. First, the xenogeneic 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, interleukin-4 and interleukin-12 for increasing the intensity of the immune response. The vaccine composition may be in the form of a xenogeneic 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 xenogeneic differentiation antigen are administered in amounts which are effective to stimulate an immune response to the target differentiation antigen in the subject. The preferred amount to be administered will depend on the species of the subject 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 xenogeneic 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 xenogeneic differentiation antigen is expressed by the subject. 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 and the antigen-coding 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 μg 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.
Once tolerance is broken through the administration of the xenogeneic differentiation antigen, subsequent treatments with syngeneic differentiation may be employed to maintain and in some cases enhance the immune response. (See, Weber, et al., “Tumor immunity and autoimmunity induced by immunization with homologous DNA.” J Clin Invest 102 (6):1258 (1998).) Thus, in one embodiment of the invention, the subject is first treated by administration of a xenogeneic differentiation antigen (for example for three treatment cycles), and subsequently by administration of a syngeneic differentiation antigen (for example for an additional three treatment cycles). As an alternative to treatment cycles using different therapeutic agents, one can use a single therapeutic agent containing both xenogeneic and syngeneic differentiation antigens. Thus, for example, a mixture of the rHer2-pING and hHer2-pING vectors, or a single vector encoding both rat and human Her2/neu under the control of a promoter such that they are expressed in a canine subject can be employed for the treatment of mammary gland tumor in canines. Vectors are available commercially, for example from Stratagene and other companies, which can express two independent genes. Commonly, these vectors use an internal ribosomal entry site, or IRES, between the two genes. This approach has the advantage of requiring approval for only a single therapeutic agent.
All documents cited herein are herein incorporated by reference in their entirety.
The invention will now be further described with reference to the following, non-limiting examples.
Example 1—Her2/neu Expression Plasmid Construction
The extracellular domain of rat HER2/neu (nucleotides 17-3799 of SEQ ID NO:1) was amplified by PCR from the pCMVneuNT (Amici et al., 1998) plasmid using the primers forward: 5′-CGAAGCTTACCATGGAGCTGGCGGCCTGG-3′ (SEQ ID NO:6) and reverse: 5′-CGGAATTCTTATGTCACCGGGCTGGC-3′ (SEQ ID NO:7). The HindIII-EcoRI fragment was cloned into pcDNA3.1(+) (Invitrogen, Carlsbad, Calif.; and FIG. 2). The original sequence of the rat neu cDNA was described previously (Bargmann et al., 1986), and is herein set forth in SEQ ID NO:1, with the coding sequence from nucleotides 17 to 3799. The rat HER2/neu coding sequence was then subcloned into the pING vector (Bergman et al., Clin Cancer Res, 9: 1284-1290, 2003, backbone depicted in FIG. 3; map depicted in FIG. 3A; and sequence as set forth in SEQ ID NO:5), to yield rat HER2/neu-pING.
Example 2—Immunization of Mammary Gland Tumor (MGT)-Positive Canines with pING-rHer2
In this trial, 10 dogs with MGT were enrolled and immunized with 100 μg of pING-rHer2 DNA per dose. The signalment for these dogs is set forth in Table 1 and the tumor staging is set forth in Table 2.







TABLE 1







Trial animal characteristics











Age

Weight



(yrs)
Breed
(kg)
















MGT 01
9
Yorkshire Terrier
1.75



MGT 02
13
Mixed
9.8



MGT 03
12
Yorkshire Terrier
5



MGT 04
7
Lhasa Apso
11



MGT 05
10
Maltese
3.35



MGT 06
12
Cavalier King Charles Spaniel
9



MGT 07
8
Pomeranian
2.8



MGT08
12
Maltese
3.9



MGT09
13
Pomeranian
2.7



MGT10
12
Yorkshire Terrier
3



Median
12
—
3.6

















TABLE 2







Tumor staging











Tumor size (cm)
MGT Type
Stage














MGT 01
2 × 2 × 4
Tubulopapillary carcinoma
T3N0M0



0.2 × 0.2 × 0.2



0.2 × 0.3 × 0.2



0.1 × 0.1 × 0.1



0.5 × 0.5 × 0.5



0.2 × 0.2 × 0.2



0.5 × 0.5 × 0.5


MGT 02
12 × 10 × 8
Lipid rich carcinoma
T3N0M0



5 × 3 × 1.5



1 × 1 × 1



1 × 1 × 0.5



0.5 × 0.1 × 0.1


MGT 03
5.6 × 4.8 × 4.6
Tubulopapillary carcinoma with
T3N0M0



1.8 × 1.5 × 1.2
fibroadenoma


MGT 04
4.2 × 5.6 × 2.5
Tubulopapillary carcinoma
T3N0M0


MGT 05
1.2 × 1 × 0.5
Simple adenoma
T1N0M0



1 × 1.4 × 0.5



1 × 1 × 0.4



0.5 × 0.5 × 0.5


MGT 06
10 × 4 × 3
Lipid rich carcinoma with
T3N0M0




fibroadenoma


MGT 07
1 × 1 × 1
Complex type
T1N0M0



0.5 × 0.5 × 0.5


MGT08
1 × 1 × 1
Complex type
T1N0M0



0.5 × 0.5 × 0.5


MGT09
2.5 × 2 × 1
Complex type
T1N0M0



1.5 × 2 × 1


MGT10
1 × 1 × 1
Tubulopapillary carcinoma
T1N0M0



0.5 × 0.5 × 0.5



0.1 × 0.1 × 0.1









As indicated, this group included five stage I and five stage III dogs, which all received three doses of vaccine at two week intervals. The first and second doses were administered with the VITAJETTM transdermal device and the third dose by intramuscular injection concurrent with electroporation. Vaccination was initiated following surgical removal of the MGT with concurrent ovariohysterectomy (OHE). All dogs were negative for regional lymph node and pulmonary metastasis. Disease free survival and overall survival times were calculated using day of surgery as day 0 with results presented in Table 3.







TABLE 3







Disease-free and overall survival time














Overall




WHO
Disease-free survival
survival time












Dog
Stage
recurrence
metastasis
(days)
Outcome





MGT 05
I
703
703
703
alive


MGT 07
I
669
669
669
alive


MGT 08
I
548
548
548
alive


MGT 09
I
536
536
536
alive


MGT 10
I
482
482
482
dead











Stage I Dogs
548
548
548
—












MGT 01
III
779
779
779
alive


MGT 02
III
212
182
212
dead


MGT 03
III
762
762
762
alive


MGT 04
III
575
381
720
alive w/ met


MGT 06
III
686
686
686
alive











Stage III Dogs
686
686
720
—


All Dogs Median
622
609
678









A group of 19 dogs was identified as historical control cases. All control dogs underwent surgical removal of MGT with concurrent OHE and were negative for regional lymph node and pulmonary metastasis. This group included 7 stage I, 3 stage II, and 9 stage III dogs. Disease free and overall survival times were calculated for these dogs using day of surgery as day 0. The signalment for these dogs is set forth in Table 4 and tumor staging for each dog is set forth in Table 5. Disease free and overall survival times were calculated for the control group and are presented in FIGS. 1A-1C.







TABLE 4







Control dog signalment













Age





Case Number
(yrs)
Breed
Weight (kg)














1
9403460
7
Mix
1.75


2
9404023
14
Poodle
2.5


3
9405132
14
Yorkshire
2.3


4
9409179
12
Finnish Spitz
6.8


5
9409043
14
Poodle
3.2


6
9500057
9
Lhasa Apso
6.5


7
9500890
14
Maltese
6


8
9500959
15
Cocker
14


9
923543
11
Siberian Huskies
16


10
9405082
13
Poodle
3.9


11
9505202
9
Mix
12


12
9600998
10
Maltese
4.6


13
9700451
13
Maltese
2.7


14
892285
12
Yorkshire
1.6


15
9502927
14
Maltese
3.2


16
9405356
10
Cocker
12


17
9409104
11
Maltese
3.8


18
9503957
6
Miniature Schnauzer
4


19
9404023
14
Poodle
3



Median
12

3.9
















TABLE 5







Tumor staging for control dogs












Clinical






NO.
Tumor size
MGT Type
Stage















1
9403460
6 × 6 × 7
Complex carcinoma
T3N0M0


2
9404023
3 × 3 × 3
Squamous cell carcinoma
T2N0M0


3
9405132
7 × 4 × 7
Simple or complex carcinoma
T3N0M0




2 × 2 × 2




0.3 × 0.2 × 0.2




0.5 × 0.5 × 0.5


4
9409179
13 × 12 × 12
Simple carcinoma with
T3N0M0




6 × 7 × 7
squamous cell carcinoma




1 × 1 × 1


5
9409043
3.5 × 2.x1
Tubulopapillary carcinoma
T2N0M0




3 × 1.5 × 1


6
9500057
3 × 2 × 2
Tubulopapillary carcinoma
T2N0M0




2 × 1 × 1


7
9500890
8 × 3 × 1
Simple carcinoma
T3N0M0


8
9500959
8 × 3 × 2
Adenocarcinoma
T3N0M0




2 × 1 × 0.5


9
923543
5 × 5 × 4
Simple carcinoma
T3N0M0




0.2 × 0.2 × 0.2


10
9405082
5 × 4 × 3.5
Simple carcinoma
T3N0M0




3 × 3.5 × 3


11
9505202
0.3 × 0.3 × 0.3
Tubulopapillary carcinoma
T1N0M0




1 × 1 × 0.5




0.4 × 0.4 × 0.4


12
9600998
0.5 × 0.5 × 0.4
Carcinoma
T1N0M0




1 × 0.5 × 0.5


13
9700451
1 × 1 × 1
Tubulopapillary carcinoma
T1N0M0




1 × 1 × 1


14
892285
0.5 × 0.8 × 0.3
Carcinoma in benign mixed
T1N0M0




1 × 0.8 × 0.5
tumor


15
9502927
5 × 4 × 4
Carcinoma in benign mixed
T3N0M0




0.5 × 0.5 × 0.5
tumor


16
9405356
10 × 3 × 1.5
Tubulopapillary carcinoma
T3N0M0


17
9409104
1 × 1 × 1
Adenocarcinoma
T1N0M0




0.5 × 0.5 × 0.5




2 × 2 × 2


18
9503957
2 × 2 × 2
Adenocarcinoma, complex
T1N0M0




0.3 × 0.3 × 0.3
type


19
9404023
2 × 2 × 1
Adenocarcinoma,
T1N0M0









Philibert et al. (2003) reviewed survival statistics for 97 dogs with MGT and reported median survival times for 41 dogs with MGT less than 3 cm in diameter to be 22 months ( ̃666 days) versus 14 months ( ̃424 days) for 56 dogs with MGT greater than 3 cm in diameter. In the absence of lymph node involvement or metastasis, tumor size less than 3 cm correlates with stage I disease and greater than 3 cm correlates with stage II or higher disease status. They did not find a difference in survival time for dogs in stages II, III or IV.
Overall median survival time for all dogs treated with the pING-rHer2 vaccine is 678 days. This was significantly higher as compared to the historical data from the 19 dogs provided by NTU indicating a median overall survival time of 300 days, and to the data published by Philibert et al. (2003) indicating 424 days overall survival time for dogs with stage II or greater MGT.
The pING-rHer2 DNA vaccine will target dogs and cats with tumors shown to over express the Her2 antigen based upon tumor tissue analysis using existing Her2 tissue expression assays. The vaccine will be administered using the VetjetTM transdermal device to deliver 100 μg of DNA into the medial thigh of dogs or lateral thigh of cats, at two week intervals for four doses. Dogs and cats that survive will receive a booster dose every six months.
The invention will now be described by the following non-limiting claims.

## Claims

1. A method for treating canine mammary carcinoma/tumor in a dog suffering from a canine mammary carcinoma/tumor comprising cells expressing a xenogeneic mammary gland-associated differentiation antigen, comprising:
a) surgically debulking the mammary carcinoma/tumor; and
b) administering to said dog a clinically-effective amount of a first nucleic acid molecule comprising a DNA sequence encoding a xenogeneic mammary gland-associated differentiation antigen under the control of a promoter which promotes expression of said xenogeneic mammary gland-associated differentiation antigen in said dog; and
wherein canines receiving said nucleic acid molecule exhibit significantly increased overall, disease-free, and/or metastasis-free survival times relative to canines receiving the surgical debulking without subsequent administration of said nucleic acid molecule, thereby treating said carcinoma/tumor.

2. The method of claim 1, wherein said nucleic acid molecule is a plasmid.

3. The method of claim 2, wherein said differentiation antigen is a canine Her2/neu antigen, and wherein said xenogeneic antigen is a non-canine Her2/neu antigen.

4. The method of claim 3, wherein said xenogeneic Her2/neu antigen is a rat Her2/neu antigen.

5. The method of claim 4, wherein said plasmid comprises nucleotides 106-3882 of the sequence as set forth in SEQ ID NO: 1, or wherein said plasmid comprises nucleotides encoding the same peptide sequence as nucleotides 106-3882 of the sequence as set forth in SEQ ID NO: 1.

6. The method of claim 1, comprising the steps of:
1) surgically debulking a canine mammary tumor/carcinoma expressing a Her2/neu differentiation antigen;
2) administering a first nucleic acid molecule encoding a xenogeneic Her2/neu; and
3) administering via electrotransfer/electroporation a second nucleic acid molecule; wherein the second nucleic acid molecule is either identical to said first nucleic acid molecule, or is a second, distinct nucleic acid molecule capable of expressing in vivo in a canine a different xenogeneic Her2/neu antigen, including those encoded by SEQ ID NOs:1, 3 or 4, or is a recombinant vector capable of expressing in vivo in said canine any Her2/neu protein, which is capable of eliciting a therapeutically effective immune response against heterologous Her2/neu expressed by said canine mammary tumor/carcinoma.

7. The method of claim 6, wherein the first nucleic acid molecule is a plasmid.

8. The method of claim 6, wherein:
1) the first administration consists of administering to said canine said first nucleic acid molecule without a needle;
2) the first nucleic acid molecule is capable of expressing in vivo in a canine a sequence comprising or consisting of the same sequence as set forth in SEQ ID NO:2; and
3) the second administration comprises or consists of administering said nucleic acid molecule of step 2.

9. The method of claim 8, wherein said first nucleic acid molecule is a plasmid.

10. The method of claim 6, wherein said second administration is provided to surviving canines once every about 3 to about 6 months.

11. The method of claim 8, wherein said second administration is provided to surviving canines once every about 3 to about 6 months.

12. The method of claim 1, which is performed about concurrently with resection of a mammary gland tumor (MGT).

13. The method of claim 4, which is performed about concurrently with resection of a mammary gland tumor (MGT).

14. The method of claim 1, wherein the administration is performed using a needle-free delivery device.

15. The method of claim 1, further comprising at least a second administration, provided at least once or at regular intervals after the initial antigen administration.

16. The method of claim 1, wherein:
a) the mean overall survival time is at least about 100 days greater;
b) the average disease-free survival time is at least about 200 days greater, and/or
c) the metastasis-free survival time is at least about 200 days greater in said canines receiving said nucleic acid molecule, relative to said canines receiving the surgical debulking without subsequent administration of said nucleic acid molecule.

17. The method of claim 16, wherein the three survival times are at least about 100, about 200, and about 200 days greater in the immunized canines relative to said canines receiving the surgical debulking without subsequent administration of said nucleic acid molecule, respectively.

18. The method of claim 9, wherein:
a) the mean overall survival time is at least about 100 days greater;
b) the average disease-free survival time is at least about 200 days greater, and/or
c) the metastasis-free survival time is at least about 200 days greater in said canines receiving said nucleic acid molecule, relative to said canines receiving the surgical debulking without subsequent administration of said first nucleic acid molecule.

19. The method of claim 18, wherein the three survival times are at least about 100, about 200, and about 200 days greater in the immunized canines relative to said canines receiving the surgical debulking without subsequent administration of said first nucleic acid molecule, respectively.