JP2002514573A - Compositions and methods for active vaccination - Google Patents

Abstract

  (57) [Summary] Non-Hodgkin's disease lymphoma (NHL) is not an anti-CD20 monoclonal antibody, but rather an immunogenic fragment of CD20 itself or its extracellular portion coupled to an antigenic carrier residue, such as keyhole limpet hemocyanin (KLH). It is treated by or administered with it. This results in a stimulation of the production of polyclonal antibodies against CD20 (or immunogenic fragments), which has the effect of reducing the number of B-cells, including malignant B-cells, and thus provides an active vaccine. The same approach applies to other diseases and conditions where the target cell has a transmembrane protein and administration of antibodies to the membrane protein or peptide has been shown to provide therapeutic benefit, and in particular, to target the growth or function of the cell. It can be used in therapy for diseases and conditions where important signals can also be transformed or received (ie, active immunotherapy). It is also known, for example, as Her2 / neu, VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2-receptor, interleukin-4-receptor and multiple drug resistance proteins P-glycoprotein is included.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD This application relates to an active Kuching approach to the treatment of cancer and other diseases. While this approach is applicable to many cancers and diseases, a preferred embodiment provides an active vaccine for the treatment of B cell non-Hodgkin's disease lymphoma (NHL).

BACKGROUND OF THE INVENTION NHL is characterized by clonal expansion of malignant B cells. A broad spectrum of patients
Although treatment of NHL has not been tested, a number of therapeutic approaches have been proposed and attempted.

[0003] The most common therapeutic approach currently used is chemotherapy. Although chemotherapy is effective for a period of time in most patients, a significant proportion of patients are incurable and have experienced a relapse.

[0004] Anti-idiotypic antibody-based treatments have been proposed. In anti-idiotype therapy, patient-specific antibodies are created using cell surface molecules that are expressed by malignant cells but not normal cells, and are then administered to patients (Mill
er et al., New Engl. J. Med. 306: 517-522, 1982). Self-idiotypic proteins from patients have also been conjugated to keyhole limpet hemocyanin to produce vaccines that have shown efficacy and can elicit B and T cell immune responses (Kwak et al.
,, New Engl. J. Med. 327: 1209-1215, 1992). Hybridoma-derived idiotypes, when reinjected into patients, act as antigen presenters and were co-cultured with patient-derived dendritic cells showing clinical efficacy (Hsu et al., Nature Medicine 2: 52-58, 1
996). Idiotype vaccines produced in lipid-based carriers are disclosed in WO 98/14170.

[0005] Treatments using antibodies directed against CD20, a transmembrane protein expressed by both normal and malignant B cells during the B cell proliferation cycle, have also been proposed. Using a single dose infusion of anti-CD20 monoclonal antibody, partial or minimal tumor regression was observed in 6 of 15 patients in phase I clinical trials (M
aloney et al., Blood 84: 2457-2466, 1994). In Phase II trials, 1 in 37 patients
Seven cases showed complete or partial regression. In December 1997, the FDA approved the first treatment based on antibodies to NHL. Rituximab (Ritvaxan, IDEC / Genentech
) Is a chimeric human / mouse antibody approved for the treatment of patients with relapsed or refractory lower or vesicular CD20 ⁺ B cell NHL (Maloney et al., Blood 90: 2188-2195, 19
97).

[0006] It has been reported that the combination of chemotherapy and anti-CD20 therapy showed a better therapeutic effect, with complete or partial remission in 11 of 11 patients (
Czuczman et al., Abstract 53, Ann. Oncol. 7, Supp. 1:56, 1996).

[0007] While therapeutic criteria using the anti-CD20 concept can be quite effective, all of these treatments have the disadvantage that they are passive therapies, ie they do not directly involve the patient's immune system. Therefore, these treatments require continuous administration of the therapeutic agent to be effective and do not provide long-term protection against relapse. Furthermore, passive therapies are monoclonal in nature and therefore have the potential for escape. Therefore, it is desirable to have an active therapy that is a therapeutic agent that, when administered to a patient, stimulates the immune response to CD20 found on B cells.

[0008] It is an object of the present invention to provide such a treatment. It is a further object of the present invention to provide an active polyclonal therapy which is difficult to avoid.

According to the present invention, NHL is administered in conjunction with or with an antigenic carrier residue such as keyhole inpet hemocyanin (KLH) rather than administration of a CD20 monoclonal antibody. It is treated by administration of an immunogenic fragment of CD20 itself or its extracellular component. This results in a stimulation of the production of polyclonal antibodies against CD20 (or an immunogenic fragment thereof) which has the effect of reducing the number of B cells, including malignant B cells. That is, the present invention provides an active vaccine. The same approach can be used to treat other diseases and conditions where the target cells have a transmembrane protein, including administering antibodies to the transmembrane protein or peptide (
That is, it is particularly applicable where diseases and conditions for which active therapy has been shown to provide therapeutic benefit, and especially where the target is capable of transducing or receiving signals important for cell growth or function. This includes, for example, Her2 / neu,
Includes VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2 receptor, interleukin-4 receptor, and P-glycoprotein, also known as multidrug resistance protein.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides active vaccine therapies that can be used to treat a variety of cancers and related conditions where it is desired to cause killing of a target cell population. The humoral immune response (antibody that recognizes target cells) alone is not considered to be sufficient to achieve the desired outcome of cell death. Immunotherapy targeted cells have been sought. The present invention differs from this conventional wisdom,
It effectively exploits the humoral immune response to target cells to provide therapeutic benefit. Therapeutic targets include cell surface proteins when bound by a ligand signal to the cell. Vaccines that elicit an antibody response mimic ligand binding and cause similar signaling phenomena that can mimic the process of programmed cell death (apoptosis), or stop cell growth or kill cancer cells. Varies the sensitivity to chemotherapy.

By way of example, the present invention is suitably used for the treatment of NHL and B cell diseases such as chronic lymphocytic leukemia, autoimmune diseases, and B cell regulatory diseases. According to this embodiment of the present invention, a peptide antigen containing at least the immunogenic portion of the extracellular domain of CD20 is prepared and coupled to or administered with an antigenic carrier protein. The CD20 component of the peptide antigen may be the same or different. Thus, for example, human patients can be treated with a peptide vaccine containing a human or mouse CD20-fragment. There is evidence that a strong immune response is elicited against heterologous proteins (Naftzger et al., Proc. Natl. Acad. Sci. USA 93: 14809-14814).
International Patent Application No. PCT / US97 / 22669, filed December 10, 1997. Incorporated herein by reference). A suitable fragment is a 44 amino acid peptide spanning amino acids 136-179 of the mouse or human CD20 sequence (SEQ ID NOS: 1 and 2). Other immunogenic fragments from the extracellular domain of CD20 or whole CD20 molecules can also be used. SEQ ID NOs: 3 and 4 are shown in Tedder et al. (J. Immunol. 142
: 260-2568, 1989) shows the nucleic acid and amino acid sequences of exon IV (extracellular domain) of human CD20, respectively.

The term “immunogenic fragment” as used herein and in the claims, refers to the administration of an immunogenic fragment linked to or administered with a carrier antigenic protein effective to break tolerance. And a molecule that includes at least a portion of the extracellular domain of a transmembrane protein that, when administered with an adjuvant, directs an immune response to the transmembrane protein. It is not required that the immunogenic fragment alone be effective to stimulate an immune response, but such stimulated fragments are not outside the scope of the invention.

A preferred antigenic carrier protein is keyhole limpet hemocyanin which can be coupled to peptides using the techniques described in the Pierce catalog protocol. Other antigenic carrier proteins used in the present invention and which can be used to disrupt tolerance include immunoglobulins, tuberculin, tetanus toxin and other proteins well known in the art.

[0014] Peptide antigens and antigenic carrier proteins, including the CD20 component, are formulated with a pharmaceutically acceptable adjuvant in a liquid carrier and administered to a patient afflicted with NHL or other B-cell disease. The compositions will generally be administered by injection, for example, intramuscularly, subcutaneously or transdermally, but also by a DNA vaccine (see US Pat. No. 5,580,859, incorporated herein by reference) or a viral vaccine.
Alternatively, it can be administered after being mixed with antigen presenting cells (APC) such as dendritic cells ex vivo. Alternatively, the antigen can be administered by injection, without adjuvant, to a host that has been previously or concurrently injected with an immune adjuvant. The particular amount to be administered to a patient can be determined by monitoring the titer of anti-CD20 antibodies generated in the patient, or by means of the average group of patients using well-known techniques.

When a peptide of the extracellular domain of human or mouse CD20 is coupled to KLH and administered to mice with an adjuvant, antibodies that react with CD20 are found in plasma (FIGS. 1A and B). These antibodies bind to Raji cells of the human lymphoma cell line, indicating that the antibodies bind to CD20 expressing cells (FIGS. 2A and B
). In addition, the number of CD19 ⁺ B cells present in mice injected with either of the two CD20-KLH conjugates is substantially reduced (-30% reduction compared to controls) (Figures 3 and 9). B
CD also expressed on immature B cell is a ^- assays using the quantitative depletion of cells CD20
19 is detected. Thus, a 30% depletion actually down-regulates the efficacy of the vaccine on CD20 ⁺ B cells.

Antibodies raised in mice after vaccination with CD20 fragments from humans or mice are specific for the peptides used, but can still elicit immunity to the corresponding peptides from other species ( 6A-D). In most cases, studies have shown that peptides, carrier proteins and adjuvants are all required for an optimal response, but some responses are detected without all of their components (FIGS. 7A-D). Several different adjuvants have also been tested and QS21
Was found to be the most effective of the adjuvants tested (FIGS. 8A-D).

While not wishing to be bound by any particular mechanism, it is believed that the vaccines of the present invention are effective via at least two routes. First, the generation of a humoral immune response to CD20 is effective in reducing the number of B cells bearing the CD20 antigen to some extent in a manner consistent with a normal immunological response to the target antigen. However, in addition to that C
Since D20 has a signaling function, binding of an antibody to the CD20 residue activates this signaling function and induces apoptotic cell death. It has been shown that such apoptotic stimulation occurs in vitro following passive treatment with a chimeric anti-CD20 antibody (Maloney et al., Blood 88 (Supp. 1): 637a, 1996).
).

[0018] T cell-mediated effector mechanisms may also be involved in the immune response. To prove this, we exemplify in Table 1 the mouse and human peptide sequences that can bind to the corresponding mouse and human histocompatibility antigens. This information is available for mouse and human
NIH Bioinformatics and Molecular Analysis Sectio using CD20 amino acid sequence
n was derived from a search of the HLA Binding Predictions database (Parker
Et al., J. Immunol. 152: 163, 1994).

Although the method of the invention has been exemplified herein using CD20 or a CD20-derived peptide as an antigen against target B cells, the invention is not limited to this embodiment. Rather, the present invention relates to a vaccine composition comprising an immunogenic portion of a transmembrane protein or peptide, particularly an extracellular domain of a transmembrane protein or peptide having a signaling function, and an antigenic protein, And / or use of a vaccine composition administered with an adjuvant that disrupts tolerability.

A non-limiting example of another transmembrane protein that can be used in whole or in part in the method of the invention is Her-2 / neu. The Her-2 / neu oncogene is a receptor-like tyrosine kinase that is expressed on the cell surface in an important part of solid tumors. Patients with early stage lung cancer have been shown to have high titers of antibodies against Her-2 / neu (Disis et al., J. Clin
Oncol. 15: 3363-3367, 19967). The amino acid sequence and domain structure of human Her-2 / neu are shown in SEQ ID NO: 5 and FIG. 4, and the isolation and expression of the extracellular domain is disclosed in International Patent Publication WO90 / 14357. This description is incorporated herein by reference. There is clinical data showing the efficacy of monoclonal antibodies to Her-2-neu in treating patients with Her-2 / neu + tumors, which may indicate a potential synergistic effect with chemotherapy. That is, according to the present invention, a vaccine composition wherein at least the immunogenic portion of the extracellular domain of Her-2-neu (amino acids 22-652) is bound to or administered with an antigenic protein or peptide such as KLH However, it can be used as a vaccine that provides the same therapeutic benefits when used in an active approach as opposed to a passive approach.

A further non-limiting example of a transmembrane protein that can be used in whole or in part in the method of the invention is the epidermal growth factor receptor (EGFR). The amino acid sequence and domain structure of human EGFR are shown in SEQ ID NO: 6 and FIG. There are significant data indicating that antibodies to EGFR may have anti-tumor activity in breast and prostate cancer, as well as in some cervical head tumors (Prewett et al., J. Immunoother. Emphasis Tumor Humoral 19: 417).
-27, 1996). The mechanism by which antibody therapy to EGFR is effective may be due to the down-regulation of endothelial growth factor production by tumor cells and thereby the ability to reduce angiogenesis (Petit et al., Am. J. Pathol. 151 : 1523-30, 1997). According to the present invention, a vaccine composition wherein at least the immunogenic portion of the extracellular domain of EGFR (amino acids 25-645) is conjugated to or administered with an antigenic protein or peptide, eg, KLH, comprises a passive approach. Can be used as a vaccine that is used in the reverse active approach to provide the same therapeutic benefit. Preferred immunogenic peptides are selected from non-deleted regions in various types of partially truncated EGFR mutants associated with some cancers.

A further non-limiting example of a transmembrane protein that can be used in whole or in part in the method of the invention is the VEGF receptor. Antibodies to VEGF receptors inhibit angiogenesis,
Therefore, the progress of the tumor can be stopped. According to the present invention, a vaccine composition that binds or administers at least the immunogenic portion of the extracellular domain of the VEGF receptor to an antigenic protein or peptide, such as KLH, is active, as opposed to the passive approach. It can be used as a vaccine that is used in the approach to provide the same therapeutic benefit.

Yet another non-limiting example of a transmembrane protein that can be used in whole or in part in the method of the invention is the IL-2 receptor. The IL-2 receptor is expressed on most T cell malignancies, and there is data indicating that antibodies against the IL-2 receptor can be used to treat T cell malignancies and autoimmune diseases. In the present invention, the composition comprises binding at least the immunogenic portion of the extracellular domain of the IL-2 receptor (eg, P55 or P75) to an antigenic carrier protein or peptide, eg, KLH, or administering it together with a vaccine. Can be used as

The vaccine compositions of the present invention can be used alone or in combination (simultaneously or sequentially) with a drug or chemotherapy that provides a therapeutic benefit to the antibody to be treated. In the case of NHL, suitable chemotherapeutic agents that can be used in combination with a CD20-based vaccine include anthrocyclidine, cis-platinum, fludarabine, corticosteroids and vinca alkaloids. These are the same chemotherapeutic agents used in combination with other vaccine compositions for other types of cancer.

Examples Example 1 A 44 amino acid fragment of the extracellular domain of human and mouse CD20 (amino acids
136-179, SEQ ID NOs: 1 and 2) were synthesized using a solid phase FMOC peptide synthesizer and coupled to KLH using the method described in the Pierce catalog protocol. The peptide coupled to KLH was then formulated for injection by formulating with QS-21 adjuvant. Balb / c mice were injected on days 1, 8, 15, 22, and 50 of the experiment according to one of the following protocols. A. B. Mouse CD20 fragment-KLH and QS-21 adjuvant B. Human CD20 fragment-KLH and QS-21 adjuvant C. KLH and QS-21 adjuvant QS-21 adjuvant P190 (unrelated protein) coupled to KLH and QS-21 B3A2 (irrelevant protein) and QS-21 animals coupled to KLH were sacrificed on day 62 of the experiment.

[0027] Serum samples from mice were diluted 1: 200 and human CD20, mouse CD20 and KLH
Antibodies that bind to were evaluated by BSA-block ELISA using goat-anti-mouse antibodies conjugated to alkaline phosphatase. As shown in FIGS. 1A and B, KL
Human CD20 coupled to H (Figure 1A) or mouse CD20 coupled to KLH
Administration of heterologous antibodies to mice injected with (Fig. 1B) produced significant polyclonal antibodies to both human and mouse CD20, but the response following administration of alloantibodies was primarily limited to antibodies to allotypes of CD20. Had been. Thus, either heterologous or homologous peptides can be used to generate an immune response.

To confirm the ability of the antibody to bind to B cells, Raji cells (a type of human B-cell lymphoma that expresses CD20 on their surface) were blocked with human IgG, washed, and the P- 1: 1 of plasma from mice vaccinated with 190-KLH control or huCD20-KLH
Incubated with 0 dilution for 30 minutes. Raji cells were incubated with B1 antibody as a positive control or with IgG2 as an isotype negative control. After washing, cells were incubated with goat-anti-mouse antibody, washed and fixed with 1% paraformaldehyde. Flow cytometry was performed in a Becton-Dickinson FACScan. The results are shown in FIGS. 2A and 3B, where the shaded dataset is the experimental dataset and the outlined dataset is the negative control. As is evident, stronger binding between the mouse antibody and Raji cells is observed as compared to that observed with the B1 antibody.

Example 2 To determine the number of B cells present in vaccinated mice, cells expressing CD19, a standard B cell phenotypic marker, were evaluated. Spleens were harvested from the animals vaccinated in Example 1 and made into a single cell suspension. After counting the total number of cells, cells were stained with FITC-labeled anti-mouse CD19 and samples were analyzed by FACScan by flow cytometry. Results of 10,000 cases were collected. The control gate was subtracted from the percentage of CD19 positive cells and multiplied by the total number of cells to determine the number of CD19 positive cells in mice treated with mouse and human CD20 peptide conjugates, and an irrelevant P190 peptide conjugate control.

As shown in FIG. 3, the absolute number of CD19 positive cells was significantly reduced in mice treated with any of the CD20 peptide conjugates. The level of CD19-positive cells is equal to the CD in the sample.
The number of 20 positive B cells is a reflection, and the number of immature CD19 ⁺ is a reflection of the CD20 ^- B cells. However, since CD19 is expressed on B cell progenitors prior to the expression of CD20, the absolute number of CD19 ⁺ B cells actually down-regulates the therapeutic effect of treatments for CD20 ⁺ B cell depletion.

Example 3 Mice were injected five times with one of the following four treatment protocols over a two month period. Human CD20 (44 amino acid fragment) -KLH + QS1 Human CD20 (44 amino acid fragment) -KLH Human CD20 (44 amino acid fragment) + QS21 KLH + QS21 Blood was collected at week 9 for analysis by ELISA. Sera from vaccinated mice were diluted 1: 200 and incubated on BSA block plates coated with msCD20, huCD20, P190 or KLH. Secondary goat conjugated to alkaline phosphatase
Add anti-mouse antibody and change color of p-nitrophenyl phosphate substrate to 405 nm
Was measured. The results are summarized in FIGS. 7A-D. In most cases, the peptide, carrier protein and adjuvant were all required for an optimal response, but some responses were detected without all components.

Example 4 Four treatment protocols: human CD20 (44 amino acid fragment) -KLH + QS21 adjuvant, mouse CD20 (44 amino acid fragment) -KLH + QS21, P190 (irrelevant protein) -KLH + QS21 and KLH and QS21 Mice were immunized according to the schedule of Example 3 using one of the mice alone. A mouse serum sample was prepared using msCD20,
The presence of antibodies reactive with huCD20, P190 and KLH was assessed by ELISA.
The results are shown in FIGS. 6A-D. After vaccination with CD20 fragments from humans or mice, the antibodies raised in the mice were specific for the peptides used, but were still able to elicit immunity to the corresponding peptides from other species.

Example 5 Mice were vaccinated five times with the huCD20 fragment-KLH conjugate without adjuvant or in combination with one of three adjuvants: QS21, BCG or Alum over a two month period. Serum samples from vaccinated mice were tested by ELISA. The results are summarized in FIGS. 8A-D. QS21 was found to be the most effective tested.

Example 6 To confirm the observations of Example 2, nucleated nucleated cells from mice vaccinated with CD20-KLH conjugate (human or mouse) in the presence of QS21 adjuvant by density gradient centrifugation. The spleen cells were collected. 1 × 10 ⁶ cells from each mouse were incubated with 2 μg of rat anti-mouse CD19 FITC-labeled antibody or an isotopically matched FITC-labeled rat antibody. Cells were washed, fixed, and analyzed by Becton-Dickinson FACScan cytometry. Figures 9A-C, DF and GI are the results of three mice from each vaccinated group. The decrease in peak reflecting the level of CD19 positive spleen cells in each mouse is evident. Table 1 Peptide sequence HLA molecule Score (T1 _{/ 2} of dissociation of molecule containing this subsequence) Human KFIRaHTPYI Kd 1600 Human FIRAHTPYI Kd 48 Human FLKMeSLNFI A_0201 21.2 Mouse HFLKmRRLEL Kd 1600 Mouse IYDCePSNSS Kd 60 Mouse LIQTSKPYV E_ A_0201 28.7

[Sequence list]

[Brief description of the drawings]

FIG. 1A and B show the results of ELISA for the formation of antibodies to human and mouse CD20 in vaccinated mice.

FIGS. 2A and B show the results for binding of control B1 antibody, or antibody in plasma from mice treated with human CD20-KLH conjugate to Raji B NHL cells.

FIG. 3 shows the levels of CP19 ⁺ B cells in mice treated with human or mouse CD20-KLH conjugate.

FIG. 4 shows the domain structure of human Her2.

FIG. 5 shows the domain structure of human EGFR.

FIGS. 6A-D show the cross-reactivity of antibodies generated in response to human or mouse CD20 fragments.

FIGS. 7A-D show the importance of carrier proteins and adjuvants in generating an immune response.

FIGS. 8A-D show immune responses generated using different adjuvants.

FIGS. 9A-I show CD19 ^- B cell levels in mice treated with human or mouse CD20-KLH conjugate.

────────────────────────────────────────────────── ─── Continued on the front page (72) Roberts, Wendy, Inventor, USA New York, New York, York Avenue 1233 (72) Inventor, Zelenetz, Andrew, Di USA, New York, Larchmont, Mohegan Road 31F Term (Reference) 4C085 AA03 AA21 BB07 BB18 BB24 EE06 EE10 FF13 FF14 FF24

Claims (20)

[Claims] 1. A method according to claim 1, wherein the patient is conjugated to a carrier protein effective to disrupt tolerance to the transmembrane protein, or at least an immunogenic portion of the extracellular domain of the transmembrane protein or a heterologous homolog thereof, and A method of active vaccination of autologous cells expressing a transmembrane protein, comprising administering a vaccine composition comprising a chemically acceptable adjuvant. 2. The transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2 receptor, interleukin-4 receptor and P-glycoprotein. The method according to claim 1, which is selected. 3. The method according to claim 1, wherein the transmembrane protein is CD20. 4. The method according to claim 1, wherein the vaccine composition comprises a peptide having the sequence given by SEQ ID NO: 1 or 2. 5. The method according to claim 1, wherein the carrier protein is keyhole inpet hemocyanin. 6. The transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2 receptor, interleukin-4 receptor and P-glycoprotein. The method according to claim 5, which is selected. 7. The method according to claim 5, wherein the transmembrane protein is CD20. 8. The method according to claim 7, wherein the vaccine composition comprises a peptide having the sequence given by SEQ ID NO: 1 or 2. 9. A patient, wherein at least the immunogenic portion of the extracellular domain of CD20 or a heterologous homolog thereof is bound to or associated with a carrier protein effective to disrupt tolerance to the transmembrane protein, and pharmaceutically A method for active vaccination of CD20 expressing B cells comprising administering a vaccine composition comprising an acceptable adjuvant. 10. The method according to claim 9, wherein the carrier protein is keyhole inpet hemocyanin. 11. The method according to claim 9, wherein the vaccine composition comprises a peptide having the sequence given by SEQ ID NO: 1 or 2. 12. A patient suffering from B-cell non-Hodgkin's disease lymphoma, wherein at least the immunogenicity of the extracellular domain of CD20 is linked to or associated with a carrier protein effective to disrupt tolerance to transmembrane proteins. A method for treating B-cell non-Hodgkin's disease lymphoma comprising administering a vaccine composition comprising a moiety, or a heterologous homolog thereof, and a pharmaceutically acceptable adjuvant. 13. At least an immunogenic portion of the extracellular domain of the transmembrane protein or a heterologous homologue thereof, and / or a pharmaceutically acceptable carrier, coupled to or with a carrier protein effective to disrupt tolerance to the transmembrane protein. A vaccine composition comprising an adjuvant to be used. 14. The transmembrane protein is a group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2 receptor, interleukin-4 receptor, and P-glycoprotein. 14. The composition according to claim 13, which is selected from the group consisting of: 15. The method according to claim 13, wherein the transmembrane protein is CD20. 16. The composition according to claim 15, wherein the vaccine composition comprises a peptide having the sequence given by SEQ ID NO: 1 or 2. 17. The kit according to claim 13, wherein the carrier protein is keyhole inpet hemocyanin. 18. The transmembrane protein is a group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, CD19 molecule, interleukin-2 receptor, interleukin-4 receptor, and P-glycoprotein. 18. The composition according to claim 17, wherein the composition is selected from the group consisting of: 19. The method according to claim 17, wherein the transmembrane protein is CD20. 20. The composition according to claim 19, wherein the vaccine composition comprises a peptide having the sequence provided by SEQ ID NO: 1 or 2.