CA2330212A1 - Compositions and methods for active vaccination - Google Patents

Abstract

Non-Hodgkin's lymphoma (NHL) is treated, not by administration of an anti-CD20 monoclonal antibody, but by the administration of CD20 itself, or an immunogenic fragment of the extracellular portion thereof, coupled to or administered with an antigenic carrier moiety such as keyhole limpet hemocyanin (KLH). This results in the 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, and thus provides an active vaccine. The same approach can be used for therapeutics for other diseases and conditions in which target cells possess a transmembrane protein, and is particularly applicable to those diseases and conditions for which administration of antibodies to transmembrane proteins or peptides (i.e., passive therapy) have been shown to provide therapeutic benefits, and especially in the situations where the target is also capable of transducing or receiving a signal important for cell growth or function. This would include, for example, Her2/neu, VEGF receptor, epidermal growth factor receptor, the CD19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein, also known as the multidrug-resistance protein.

Description

WO 99/579$1 PCT/US99/10065 COMPOSITIONS AND METHODS FOR ACTIVE VACCINATION
BACKGROUND OF THE INVENTION
This application relates. to an active vaccine approach to the treatment of cancer and other diseases. The approach is applicable to a number of cancers and diseases, although a preferred embodiment provides an active vaccine for treatment of B
cell Non-Hodgkin's Lymphoma (NHL).
NHL is characterized b~y a clonal proliferation of malignant B cells. The treatment of NHL across a broad spectrum of patients remains a challenge, although numerous therapeutic approaches have been proposed and tried.
The most common therapeutic approach being used today is chemotherapy.
While chemotherapy is effective for some period of time in most patients, a significant percentage of patients are not cured and experience a relapse.
Treatments have been proposed based on anti-idiotype therapy. In anti-idiotype therapy, a cell surface molecule which is expressed by malignant cells but not by normal cells is used to create patient-specific antibodies which are then administered to the patient. See, Miller, et al., New Engl. J. Med. 306: 517-522 (1982}.
Autologous patient--derived idiotype proteins have also been conjugated with keyhole limpet hemocyanin to produce a vaccine which has demonstrated efficacy and can elicit B and T cell immune responses. Kwak et al., New Engl. J. .Med. 327: 1209-1215 (1992). Hybridoma-derived idiotype was co-cultured with patient-derived dendritic cells which acted as antigen presenters upon re-infusion into the patient and showed clinical efficacy. Hsu et al., Nature Medicine 2: 52-58 (1996). Idiotypic vaccines made in lipid-based earners are disclosed in International Patent Publication W098/14170.
Treatments have also been proposed using antibodies directed to CD20, a transmembrane protein that is expressed by both normal and malignant B-cells during parts of the B cell development cycle. Using single-dose infusions with anti-CD20 monoclonal antibodies, partial or minor tumor regressions were observed in 6 of 15 patients in a Phase I
clinical study. Maloney et al., Blood 84: 2457-2466 (1994). In Phase II
studies, 17 of 37 patients showed complete or partial rE;missions. In December 1997, the FDA
approved the first antibody-based therapy for NHI~. Rituximab (Ritvaxan, IDEC/Genentech) is a chimeric human/murine antibody approved for the treatment of patients with relapsed or refractory low-grade or follicular CD20+ B cell NHL. Maloney et al., Blood 90: 2188-2195 (1997).
Combinations of chemotherapy and anti-CD20 therapy have been reported as having better therapeutic efficacy, with 11 of 11 patients showing complete or partial remission. Czuczrnan et al., Abstraca 53, Ann. Oncol. 7, Supp. 1: 56 (1996).
While therapeutic regimens using anti-CD20 concepts are potentially effective, all of these therapies have the drawback of being passive therapies, i.e., they do not directly involve the immune system of the patient. Thus, these therapies may require the continued administration of the ther<ipeutic agent for efficacy and do not provide any long-terns protection against recurrence. :ln addition, the passive therapy is monoclonal in nature, therefore escape is possible. It would therefore be desirable to have an active therapy, that is a therapeutic agent which when administered to the patient stimulates an immune response against CD20 found in B-cells.
It is an object of the present invention to provide such a therapy. It is a further object of the invention to provide an active polyclonal therapy that is difficult to evade.
SUMMARY OF THE INVENTIOI\f In accordance with the present invention, NHL is treated, not by administration of an anti-CD20 monoclonal antibody, but by the administration of CD20 itself, or an immunogenic fragment of the extracellular portion thereof, coupled to or administered with an antigenic carrier moiety such as keyhole limpet hemocyanin (KLH).
This results in the stimulation of the production of polyclonal antibodies against CD20 (or an immunogenic fragment thereof) which has the affect of reducing the number of B-cells, including malignant B-cells. Thus, the invention provides an active vaccine.
The same approach can be used for therapeutics for other diseases and conditions in which target cells possess a transmembrane protein, and is particularly applicable to those diseases and conditions for which administration of antibodies to transmembrane proteins or peptides (i.e., passive therapy) have been shown to provide therapeutic benefits, and especially in the situations where the target is also capable of transducing or receiving a signal important for cell growth or function. This would include, for example, Her2/neu, VEGF
receptor, epidermal growth factor receptor, the CD 19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein, also known as the multidrug-resistance protein.
BRIEF DESCRIPTION OF THE FIGURES
Figs. lA and B show ELISA results for formation of antibodies to human and mouse CD20 in vaccinated mice;
Figs. 2A and B shoves results for binding of control B 1 antibodies or antibodies in plasma from a mouse treated with human CD20-KLH conjugate with Raji B
NHL cells;
Fig. 3 shows CP19+B cell levels in mice treated with human or mouse CD20-KLH conjugate;
Fig. 4 shows the donnain structure of human Her2;
Fig. 5 shows the domain structure of human EGFR;
Figs. 6A-D shows the cross-reactivity of antibodies generated in response to human or mouse CD20 fragments;
Figs. 7A-D show thc: importance of carrier protein and adjuvant in generating an immune response;
Figs. 8A-D shows the immune response generated using different adjuvants;
and Figs. 9A-I shows CI'19+B cell levels in mice treated with human or mouse CD20-KLH conjugate.
DETAILED DESCRIPTION OF T'HE INVENTION
The present invention provides an active vaccine therapy which can be used in the treatment of a variety of cancer's and related conditions in which it is desirable to bring about the death of a target group of cells. Conventionally, immunotherapies targeting cells have sought to obtain a cellular immune response (T-cells that recognize the target cells) , since a humorai immune response (antibodies that recognize the target cells) alone is not deemed sufficient to achieve the desired result of cell death. The present invention departs from this conventional wisdom, and effectively utilizes a humoral immune response against the target cells to provide therapeutic benefit. The targets for therapy include cell surface proteins that when bound by a ligand signal to the cell. The vaccine induced antibody response will mimic ligand binding and cause similar signaling events which can imitate the process of programmed cell death (apoptosis) or halt the cell from growing or change the cancer cell's sensitivity to chemotherapy.
By way of example, the invention is suitably employed in the treatment of NHL and other B cell diseases such as chronic lymphocytic leukemia, auto-immune disorders and B-cell regulatory disorders. In accordance with this embodiment of the invention, a peptide antigen is prepared which contains at least an immunogenic portion of the extracellular domain of CD20 coupled to or administered with an antigenic carrier protein.
The CD20 component of the peptide antigen may be syngeneic or it may be xenogeneic.
Thus, for example, human patients may be treated with a peptide vaccine containing a human or a mouse CD20-fragment. There is evidence that strong immune responses can be elicited against xenogeneic proteins. Naftz~;er et al., Proc. Natl. Acad. Sci. (USA) 93: 14809-14814 (1996); International Patent Application PCT/US97/22669, filed December 10 199?, incorporated herein by reference. A suitable fragment is the 44 amino acid peptide spanning amino acids 136 to 179 of the sequf;nce of mouse or human CD20. (Seq. ID Nos.
l and 2) Other immunogenic fragments derived from the extracellular domain of CD20, or the entire CD20 molecule may also be used. Seq. ID. Nos. 3 and 4 shows the nucleic acid and amino acid sequences, respectively, of exon VI (the extracellular domain) of human CD20 as reported by Tedder et al., J. Immun~ol. 142: 2560-2568 (1989).
As used in the specification and claims hereof, an "immunogenic fragment" is a molecule which includes at least a portion of the extracellular domain of a transmembrane protein to direct and immunologica.l response to that transmembrane protein when the immunogenic fragment is coupled to or administered with an antigenic earner protein effective to break tolerance and administered with an adjuvant. It is not required that the immunogenic fragment alone be effective to stimulate an immune response, although such stimulation would not take a given fragment outside the scope of the present invention.

-S-A preferred antigenic. carrier protein is keyhole limpet hemocyanin which can be coupled to peptides using techniques described in Pierce Catalog Protocol.
Other antigenic carrier proteins which can be used to break tolerance might be used in the invention include immunoglobulins, tuberculin, tetanus toxin and others well known in the art.
The peptide antigen containing the CD20 component and the antigenic carrier protein is formulated with a pharmaceutically acceptable adjuvant in a liquid carrier and administered to a patient suffering from NHL or another B cell disease. The composition will generally be administered by injection, for example, intramuscular, subcutaneous or intradermal injection, but might also be administered by way of a DNA vaccine (See US
Patent No. $,580,859, incorporated herein by reference) or a viral vaccine, or after mixing with antigen presenting cells (APC';s) such as dendritic cells, ex vivo.
Alternatively, the antigen may be administered without adjuvant by injection into a host prepared by prior or simultaneous injection of an immune adjuvant. Specific amounts to be administered to a patient can be determined by monitoring the titer of anti-CD20 antibodies developed by the 1 S patient, or by an average group of patients using well-known technology.
When a peptide of the extracellular domain of human or mouse CD20 is coupled to KLH and administered with an adjuvant to mice, antibodies which react with CD20 are found in plasma. (Figs. lA and B) These antibodies bind to Raji cells, a human lymphoma cell line, indicating the ability to bind to a cell expressing CD20.
(Figs. 2A and B). Moreover, the number of CDl9t B cells present in mice injected with either of the two CD20-KLH conjugates declines substantially (~30% decrease relative to controls). (Figs. 3 and 9). The assay used to quantitatc; B cell depletion detects CD 19 which is also expressed on immature B cells that are CD20-. Thus, the 30% depletion actually underestimates the efficacy of the vaccine against CD20' B cells. .
Antibodies generated in mice after vaccination with human or mouse-derived CD20 fragments are specific for the peptides used, yet are capable of inducing immunity to the corresponding peptide from other species (Figs. 6A-D). Studies showed that in most instances the peptide, carrier protein and adjuvant are all needed for optimal response, although some responses were detected using less than all of the components.
(Figs. 7A-D).

Several different adjuvants were also tested, and QS21 was found to be the most effective of those tested. (Fig. 8A-D).
While not intending to be bound by any particular mechanism, it is believed that the vaccines of the present invention are effective via at least two pathways. First, the S generation of a humoral immune response to CD20 is effective to some extent to reduce the numbers of B cells bearing CD20 antigen in a manner consistent with normal immunological response to a target antigen. In addiction, however, because CD20 has a signaling function, the binding of antibody to the CD2C1 moiety activates this signaling function to trigger apoptotic cell death. Such stimulation of apoptosis has been demonstrated to occur in vitro following passive treatments with a chimeric anti-CD20 antibody. Maloney et al., Blood 88 (Supp. 1): 637a (1996).
It is also possible that T cell mediated effector mechanisms are involved in the immune response. As evidence of this, we illustrate in Table 1 the mouse and human peptide sequences capable of binding to the corresponding mouse and human histocompatability 1 S antigens. This information was deriived from a search of the NIH
Bioinformatics and Molecular Analysis Section HLA Binding Predictions database using the mouse and human CD20 amino acid sequences. (Parke;r et al., J. Immunol. 152: 163 (1994)).
While the method of the invention is illustrated here using CD20 or CD20-derived peptides as the antigen to target B cells, the invention is not limited to this embodiment. Rather, the inventions encompasses the use of vaccine compositions comprising an immunogenic portions of the extracellular domain of transmembrane protein or peptide, particularly a transmembrane protein or peptide having signaling function, coupled to or administered with an antigenic protein and/or adjuvant to break tolerance.
A non-limiting exarr~ple of another transmembrane protein which 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 of a significant portion of solid tumors. It has been shown that patients with early stage breast cancer have a high titer of antibodies to Her-2/neu. Discs e1; al., J. Clin. Oncol. 15: 3363-3367 (19967). The amino acid sequence and domain structure: of human Her-2/neu are shown in Seq. OD.
No. 5 and Fig. 4, and isolation and expression of the extracellular domain has been disclosed.

International Patent Publication No, WO 90/14357, which is incorporated herein by reference. There is clinical data showing efficacy of monoclonal antibodies against Her-2-neu in the treatment of patients with Her-2/neu+ tumors, and potential synergism with chemotherapy. Thus, in accordance; with the present invention, a vaccine composition comprising at least an immunogenic; portion of the extracellular domain of Her-2-neu (amino acids 22 to 652) coupled to or administered with an antigenic protein or peptide such a KLH
can be used as a vaccine to provide the same therapeutic benefits using an active as opposed to a passive approach.
A further non-limiting example of a transmembrane protein which can be used in whole or in part in the method of the invention is epidermal growth factor receptor (EGFR). The amino acid sequence; and domain structure of human EGFR are shown in Seq.
ID. No. 6 and Fig. 5. There is significant data showing that antibodies to EGFR can have anti-tumor activity in breast and prostate cancer, as well as several head and neck tumors.
Prewett et al., J. Immunother. Emp~iasis Tumor Humoral 19: 419-27 (1996). The mechanism by which antibody therapy against EGFR may be efficacious can be through the ability to down-regulate vascular endothelial growth factor production by tumor cells and thereby decrease angiogenesis. Petit et al., Am. J. Pathol. 151: 1523-30 (1997). In accordance with the present invention, a vaccine composition comprising at least an immunogenic portion of the extracellular domain of EGFR I;amino acids 25 to 645) coupled to or administered with an antigenic protein or peptide such a KLH can be used as a vaccine to provide the same therapeutic benefits using an active: as opposed to a passive approach.
Preferred immunogenic peptides would be selected from regions not deleted in the various types of truncated EGFR mutants associated with some cancers.
A further non-limiting example of a transmembrane protein which can be used in whole or in part in the method of the invention is VEGF receptor. There are significant data showing that antibodies to VE?(JF receptor can inhibit angiogenesis and thereby halt tumor progression. In accordance with the present invention, a vaccine composition comprising at least an immunogenic portion of the extracellular domain of VEGF
receptor coupled to or administered with an antigenic protein or peptide such a KLH can be used as a -g_ vaccine to provide the same therapeutic benefits using an active as opposed to a passive approach.
Still a further non-limiting example of a transmembrane protein which 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-cells malignancies, and there is a data showing that antibodies to the IL-2 receptor can be used in the treatment of T-cell malignancies and autoimmune disorders. In the present invention, a composition is made comprising at least an immunogenic portion of the extrace:llular domain of the IL-2 receptor (e.g., PSS or P7S) , coupled to or administered with an antigenic carrier protein or peptide such as KLH. and used as a vaccine.
The vaccine compositions of invention can be used alone or in combination (concurrently or sequentially) with drugs or chemotherapy agents that provide therapeutic benefit for the condition being treated. In the case of NHL, suitable chemotherapy agents which can be used in combination with the CD20 based vaccine include alkylating agents, 1 S anthrocyclines, cis-platinum, fludanabine, corticosteroids and vinca alkaloids. These same chemotherapy agents which might lie used in combination with other vaccine compositions for other forms of cancer.

44 amino acid fragments of the extracellular domains of humans and murine CD20 {amino acids 136-179, Seq. l:D Nos. 1 and 2) were synthesized using a solid-phase FMOC peptide synthesizer and coupled to KLH using the methodology described in the Pierce Catalog Protocol. The peptide coupled to KLH was then prepared for injection by formulation with QS-21 adjuvant. Balb/c mice were injected according to one of the following protocols on clays 1, 8, 15, 22 and SO of the experiment:
A. Murine CD20 fragment-KLH with QS-21 adjuvant B. Human CD2.0 fragment-KLH with QS-21 adjuvant C. KLH with Q~S-21 adjuvant D. QS-21 adjuvant E. P 190 (irrelevant protein) coupled to KLH with QS-21 adjuvant F. B3A2 (irrele:vant peptide) coupled to KL,H with QS-21 adjuvant.
The animals were sacrificed on day 62 of the experiment.
Serum samples from the mice were diluted 1:200 and evaluated by BSA-blocked ELISA using goat-anti-mouse antibody conjugated to alkaline phosphatase for antibodies which bind to human CD20, mouse CD20 and KLH. As shown in Figs lA
and B, mice injected with human CD20 coupled to KLH {Fig. lA) or mouse CD20 coupled to KLH
(Fig. 1B) administration of xenoge;neic antibody produced a significant polyclonal antibody response to both human and mouse CD20, while the response following administration of syngeneic antibody was principally limited to antibodies to the syngeneic form of CD20.
Either xenogeneic or syngeneic peptide can therefore be used to generate an immune response.
To confirm the ability of the antibodies to bind to B cells, Raji cells (a form of human B-cell lymphoma that expresses CD20 on its surface) were blocked with human IgG, washed and then incubated for 30 minutes on ice with a 1:10 dilution of plasma from a mouse vaccinated with P-190-KLH control or huCD20-KLH. As a positive control, Raji cells were incubated with Bl antibody, or IgC~2 as an isotypic negative control. After washing, the cells were incubated with goat-anti-mouse antibody, washed and fixed with 1 %
paraformaldehyde.
Flow cytometry analysis was performed in a Becton-Dickinson FACScan. The results are shown in Figs 2A and 3B, wherein the shaded data set are the experimental data set and the outlined data set is the negative controls. As is apparent, there is a strong binding of mouse antibodies and Raji cells, comparable to that observed with B1 antibody .

To assess the number of B cells present in vaccinated mice, an evaluation was made of cells expressing CD19, a :standard phenotypic marker for B cells.
Spleens were harvested from the animals vaccinated in Example 1 and put into a single-cell suspension.
After counting the total number of cells, the cells were stained with FITC-labeled anti-mouse CD19 and the samples were analy~:ed by flow cytometry with a FACScan. 10,000 events were collected. The percentage of CD19 positive cells minus the control gate was multiplied by the total number of cells to determine the number of CD19 positive cells in mice treated with the mouse and human CD20 peptide conjugates, and the P190 irrelevant peptide conjugate control.
As shown in Fig. 3, t:he absolute number of CD19 positive cells was significantly reduced in mice treated with either of the CD20 peptide conjugates. The level of CD19 positive cells is a reflection of the number of CD20 positive B cells, and the number of immature CD19+, CD20- B cells in the samples. The absolute number of CD19+ B
cells actually underestimates the therapeutic efficacy of the treatment for elimination of CD20+ B
cells, however, since CD19 is expressed on B cell progenitor cells before expression of CD20.

Mice were injected five times over two months with one of four treatment protocols as follows:
human CD20 (44 as fragment)-KLH plus QS 1 human CD20 (44 as fragment)-KLH
human CD20 (44 as fragment) plus QS21 KLH plus QS21 Blood was collected on week 9 for analysis by ELISA. Sera from the vaccinated mice were diluted 1:200 and incubated on BSA blocked plates coated with msCD20, huCD20, P190 or KLH. Secondary goat anti-mouse antibody conjugated to alkaline phosphatase was added, and the color change of p-nitrophenyl phosphate substrate was measured at 405 nm. The results are summarized in Figs. 7A-D. In most instances the peptide, earner protein and adjuvant are all needed for optimal response, although some responses were detected using less than all of the components.

Mice were vaccinated according to the schedule of Example 3 using one of four treatment protocols: human CL>20 (44 as fragment)-KLH plus QS21 adjuvant, mouse CD20 {44 amino acid fragment)-KLH plus QS21, P190 (irrelevant protein)-KLH
+QS21 and KLH and QS21 alone. Mouse semm samples were evaluated by ELISA for the presence of antibodies reactive with msCD20, huCD20, P190 and KLH. The results are shown in Figs.
6A-D. Antibodies generated in mice after vaccination with human or mouse-derived CD20 fragments are specific for the peptides used, yet are capable of inducing immunity to the corresponding peptide from other species.

Mice were vaccinated five times over two months with huCD20 fragment-KLH conjugate with no adjuvant o~r in combination with one of three adjuvants:
QS'? 1, BCG
or Alum. Serum samples from the vaccinated mice were tested by ELISA. The results are summarized in Figs. 8A-D. QS21 was found to be the most effective of those tested.

To confirm the observations of Example 2, nucleated spleen cells were recovered by centrifugation in a dc;nsity gradient from mice vaccinated with a conjugate (human or mouse) in thf: presence of QS21 adjuvant. 1 X 10G cells from each mouse were incubated with 2 ,ug o~f rat anti-mouse CD19 FITC-labeled antibody or with isotope-matched FITC labeled rat antibody. Cells were washed, fixed and analyzed with a Becton Dickinson FACScaliber cytometer. Figs 9A-C, D-F and G-I show the results for three exemplary mice of each vaccination group. The decrease in the peak reflecting levels of CD19 positive spleen cells in eaclu of the mice is apparent.

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Lau pro Thr Asn n~a Ser Leu Ser phe Lw Gln Asp Tle Gln Glu Val Gln Gly Tyr Va::eu Ile Ala His Asn Gln Val Arg G1:: Val Pro Leu g5 90 95 Gir. i;rg Le;: :~r:, I_.: Val F~rg G~,: Thr G: ~ Leu Phe G:.v Asp Asn Tyr ;Ov i05 ii0 Ala Leu Ala V~:i ~e:: ~lsp Asn Gly Asp Pro Leu Asn As:: Thr Thr Pro 115 120 1 2:i Val T::r Gly .:la Se: pro Gly Gly Leu Arg Glu Leu G~ : Leu Arc Ser Leu Thr Glu :~.e Le:: :.ys Gly Gly Val Leu Ile Gln «rg Asn Pro Gin lg5 150 .155 1G0 Leu Cys Tyr Gi:". :1JJ Thr Ile Leu Trp Ly:, Asp I1 a Phe L:is Lys Asn _",, ? 7~ 175 Asn G1:~ Leu yla :,~:: Thr Leu Ile Asp Tr Asn Arg Ser Arg Ala Cys :80 135 190 :Iis Pro Cyis 5:: ?~o !~iet Cys Lys Gly Se: :?rg Cys Try, Gly Glu Ser SUSSTiTUTE SHEET (RULE 2fi) wo ms~9si pcrius~noo6s 195 200 ?v5 Ser Glu Asp C; s Gln Ser Leu Thr tt:~0 Thr Val Cys :,'.~a Gly Gly Cys 210 215 3::0 F,la .~'.~c- Cys Lys Gly Pro Leu Pro ~~~ Asp Cys Cys s Glu Gln Cys 225 . 230 235 240 r.ia aia Gly Cys Thr Gly Pro Ly:, K-s Se_ :~si~ Crr~ -.e4 Ala Cys Leu His Pile Asn ::is Ser Gl.y Ile Cys G:.',. Leu His Cys ?ro Ala Leu Val Thr Tyr Asn Thr Asp Thr Plie Glu Ser Met: Pro Asrl Pro Glu Gly Arg 275 280 . 285 T~ r Tt:r Phe Gly Ala Ser Cy Val "..._ ::la C,:~~; i~rc Ayr Asn Tyr Leu J
2~p 295 300 Ser Thr Asp V~1 Gly Ser Cys '!'hr ~~:u Val Cys i~ro Leu ciis Asn Gln Glu Vai Thr :,la Glu rlsp Gly Thr G~:. Ark Cys G1u ~ys Cys Ser Lys Pro Cys ~11a ~lr~ Val Cys Tyr Gly Lo:: GIy M~:.~ G1;: :iis Leu Arg Glu 3.0 3~i:i 350 ~~ai srr Ala Val Thr Ser Ala Asn Iie.Gln Glu Phc .~',la Gly Cys Lys Lys .le Phe Gly Ser Leu Ala Phe tee;: Pro Glu See ?::e Asp Gly Asp Pro is Ser As:: Thr Ala Pro Leu G:.:: Pro Glu Gln ~eu Gln Val Phe Glu Thr Leu Glu Glu Ile Thr Gly 'yr Leu 't'yr Ile Se: Ala Trp Pro ~i 10 .05 Asp Scr Leu :': o rap Leu Ser Val :'::~~ Gln i,sn Leu G:. : Val Ile Arg 4'2U ~:~~; 430 Giy Arg Ile Leu His Asn Gly Ala ~;~~ Ser Leu '!'hr ~eu Gln Gly Leu Gyy ;1~ Ser ~.r ~ Leu Gly Leu Arg Si:_ Leu Ar J Glu Leu Cly Ser Giy SUBSTITUTE SHEET (RULE 28) 450 455 4Go Leu t:la Leu Ile His fiis Asn Thr Ni.~ Leu Cy~ Pl7e V~1 ~iis Thr 'Jal PYO '''rp tZ:i7 Gi:l L6'u l~hC: :1'-C: flSn I~YO 1-~l:i Gli7 Alc'1 Lel: Le,::
ftl,i Thr Ala rsi: ArS Pro Glu Asp G;u Cys Val Gly Glu Gly i:eu Ala Cys His 50C 5i~5 S10 Gln Lcu Cys Air: ark Gly ais Cys 'i'rp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys See Gln Pi7c Lc:: .:,rr Gly Gln G1u Cys Vai Glu Glu Cys X30 53~ 540 ..rr VG1 Leu Gi : Gly Leu Pro Arg G~.u Tyr Vai Asn A?a yrg ais Cys Leu Pro Cy:; i:is Pro Glu Cys Gln Pro Gln r'ssn Cly See Val Thr Cys Phe Gly Pro Glu ?sla Asp Gln Cys Val A1~ Cys Ala His Tyr Lys Asp Pro Pro Phe CyVal Ala Arc3 Cys Pro Ser Gly Val Lys Pro Asp Leu Se: '.";m P:et Pro Ile Trp Ly~~ Phe Pro lisp Glu Glu Giy ::la Cys Gin Gi0 G15 G20 Pro Cys Pro Ile an Cys Thr ~:is Ser Cys Val Asp Leu Asp Asp Lys Giy Cys Pro Ala G;u Gln Arr Ala Ser Pro Leu Thr Sc:r Ile Ile Ser Ala Val Val Gly .le Leu Leu Val Val Val Leu Gly Vai Val Phe Gly .'e Leu T_1~ Lys .-.-'::g Arg Gln Gln Lys Ile Ark Lys Tyr Thr Met Arg Arn Leu Lcu Glr ~=a Thr G1~ Leu Val Glu Pro Leu T::r Pro Ser Gly ria !".et Pro :a : G:: Aia Gin idea ArJ Ilc Leu Lys Gu :'hr Glu Leu SUBSTTTUTE SHEET (RULE 26) 70~ 710 715 720 Arg Lys Val Lya Val Lcu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Iia Trn ie Pro Asp Gly Glu Flsn Val Lys Ile Pro Val Ala Iie 7d0 '1 ~5 750 Lys Val Leu Arr, Glu Asn Thr Ser Pro Lys Ala Asn :.ys Glu Ile Leu :asp Glu Ala ':yr V4i I~ct Ala Gly Val Cly Ser Pro Tyr Val Ser hrg Leu Leu Gly ile Cys Leu Thr. Ser Th: Val Gin Leu Val Thr Gln Leu tiet ?~o Tyr G;.l Cys Leu Leu Asp Nis Val elrg Clu Asn Arg Gly Arg Leu Giy Sir G_:: Asp Leu Leu Asn Tr p Cys Met Gln Ile Ala Lys Gly Met Ser Tyr LeL Glu Asp Val Arg L::u Val Fiis Arg Asp Leu Ala Ala Arc3 As:~ Val Leu Val Lys Ser Pro Asn Flis Val Lys Ile Thr Asp Phe G1}J I,e;: Ala ?~: ;, Luu Leu Asp Ile Asp Glu 'lhr Glu ':yr His Ala Asp 8G5 870 3'75 880 Gly Gly Lys Vaaro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg Phe T~° :iis Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Ple~ Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arc3 Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Th_ plc Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile ~:sp Se. Gi;: ~ys :.Yc °ro Ar J Phe ::~~ Giu Leu Val Ser Glu Phe SUBSTITUTE SHEET (RULE 26) wo ms~9si pcrius9~noo6s S~r Ar0 Met r-a yr~ rasp Pro Cln r.rJ Phe Vsl Val T_le Gln Asn Glu Asp Leu Gly Pro nla Se:r Pro Leu Asp Ser :'::_~ Nhv Tyr Arg Scr Leu Leu Glu t~sp A~:~ Asp Met Gly Asp Leu Val r?sip r'!la Glu Gl.u Tyr Leu O10 1015 20'0 Val Pro Gln C1: Gly Phe Phe Cys Pro r?sp Pro Ala Pro Gly Ala Gly Gly 1':~.t VaI ~iis :his Arch I-tis ~Ar 0 Sa: Ser Scr '1'hr Ar J Ser Gly Gly ;OqS 1050 1055 Giy :~sp L4u Thr Leu Gly Leu Glu Pro Svr Glu Glu Git; Ala Pro Arg 106"v lOGS 1070 Ser Pro Leu hia Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His A:;p Pro Scr Pro ~cu Cln Ark Tyr Scr Cl.u Asp Pro '!'hr Val Pro Leu P ro S'° Glu Tr :?sp Gl y Tyr Val T~'ia Pro Lieu '1'hr Cys Ser E'ro Gln t_ ;125 1130 1135 Pro Glu Tyr Val :>,sn Gln Pro Asp Val ~lrg Pro Cln Pro Pro Ser Pro Ar0 -Glu Cly Pro Le~,: Pro Ala Ala ArcJ 1'ro Ala Gly Aia '!'hr Leu Glu Arg Pro Lys Th= :,eu Ser Pro Gly Lys ~~~sn Gly Vai Val Lys Asp Val y170 1175 1180 Phe :via i~h~ Gly Gly Ala Val Glu As.~ Pro Glu '!'yr Leu Tl:r Pro Gln Gly Gly Ala r?la Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala .205 1210 1215 Phe ysa ~s Le:: ':.,~r Tyr Trp Asp Gin Asp Pro Pro Clu Arg Gly Ala suesn~ sHE~ ~RU~ Zs~

Pro Pro Ser ~'::r Phe Lys GIy Thr Pro Ti:r Ala G1a As : Pro Glu Tyr Leu Giy Leu As;: Val Pro Val ;250 1255 <210> 6 <211> 1210 <212> PRT
< 213 > : i i3MAN
<220>
<223> '.~.u:aan ~G:'R
<400> 6 Met :erg Pro Scr Gly~ Thr Ala Gl.y Ala i;la Leu Lcu Al.a Leu Leu Ala 1 5 7.0 15 Ala Leu Cys Pro «la Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln Gly T.::r Ser Asn Lys Leu Thr Gln Leu Giy Thr Phe Glu Asp His Phe Leu Ser Leu Gln err, Met Phe Asn Asn Cys Glu Val Vai ~eu Gly Asn Leu Gi:: Iie T'.~.r Tyr Val Gln Arg Asn Tyr Asp Leu Ser ?ne Leu Lys Thr I.e Gln Glu Vai Ala Gly Tyr Val Leu Iie Ala Lei: As:. Thr Val Glu Arg ire Pro Leu Glu Asn Leu Gln Ile Iie Arg Gly nsn Met Tyr 100 105 .10 'Pyr Giu :~s : Sw °;.~ rlia Leu Ala Val Lcu :cur A::n 'I'yr usp hla Asn Lys T:r Giy Leu Lys Glu Leu Pro Met rrg Asn Leu Gln Glu Iie Leu 1.3u 13-'i 140 His Giia Val ~=.rs Phe Sor Asn Asn Pro Ala Leu Cys ~lsn Val Glu 145 150 15:i 16G
suesmurE sHE~r ~aut.s zs~

wo s9is~9si 9 rcrius~noo6s Ser Ile Gin Trp Arg Asp Ile Val Ser 5or Asp 1'hc: LoL Ser Asn Met Ser Met Asp 1'he Gln Asn l~lis Lcu Gly Scr Cy:. Gln L:.~:: Cys Asp Pro ly ' 190 Sar Cys Pro Asn Gly Ser Cys Trp Gly ::la Gly G1U G1;: Asn Cys Gln Lys Leu Thr Ly~ ale Ile Cys Aia Gln Gln Cy~ Ser G1~% Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Alu aIa Gly Cys Thr Gly Prp Arg Giu SCr A5p CYS L~U Val <:y~ Ar J L}'S PhC Ar C ASp 4~'.5 250 255 Glu Ala Thr Cys Lys App Thr Cys p~~o Pro Leu Met Leu Ty= Asn Pro mhr mrr Tyr Gln Met App Val :?~n Pro Glu Gly Ly~ Tyr Scr Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val ArJ Aia Cys Giy Ala Asp Ser '1'yr G.u MGt Glu Glu asp Gly Vai Arc3 Lys Cys Lys Lys Cys Glu Gly Pro C}'s Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu.Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys fiis Phe Lys Asn Cys Tar Ser Ile Ser Gly Asp 360 :565 Leu His Ile Leu ?ro Val Ala Phe Arc; Gly Asp Ser Phc Thr His Thr Pro Pro Lcu Asp ?ro Gln Glu L~:u Asp Ile Lcu Lys Thr Val Lys Glu ~ c Tr G1~ pt~e :,cu Leu Ile Gln Ala Trp Pro ,Glu easn Arg Thr Asp 410 4I~
~: 0 5 suBSmur~ sHe~ cRU~ is) WO 99157981 1 ~ PCT/US99/10065 Lcu ::is Nla Phe Glu As.~. Leu Glu Ilc .ilc Arr, Gly ~rg Thr Lys Gln ~12 5 ~.ii:; Gly Cln Phc :per. Lcu ~~ia Va.l Va:. Ser i~eu As:: Ile Thr Ser Leu . i ~1:i .l~Ii~

Gly Leu ArJ Se: Lcu Lys Glu ale Scr Asp Gly lisp Val Ile Ile Ser Gly Asn Lys AS.~. ~eu Cys Tyr Ala Ann :'hr Ile Asr. Trp Lys Lys Leu Phe GIy Thr See Gly Gln Lys 'fhr Lys I1:Ile Si:r Asn Arg Gly Glu ~.S5 490 495 rlsn Ser Cys Lys .::a Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Giu Giy Cys '::i~ GlY Pro Glu Fro Arg Asp Cys Vai Ser Cys Arc,~ Asn Val Ser Arg Gly yrg Glu Cys Val Asp Lys Cys Lys Leu Leu Glu Gly Glu Pro Arg Glu ?he Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met hsn Ilc Th r Cys Thr Gly Arch Gly Pro ~G5 r:sp i~sn CyJ T_1V ,:t.~. Cys Ala His Tyr .Lle Asp Gly LLO h1s CyJ Val Lys Tl:r Cys Pro :.tea Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr rla F~sp ::la Gly His Val Cys His Leu Cys H is Pro Asn Cys Thr 'fyr Gly Cys ~ r Gly Pro Gly Lcu Giu Gly Cys i~:o Thr Asn Gly Pro Lys Ile Pro Ser I'_e Ala Thr Gly i4ct Val Gly Ala Leu Leu Leu .i 5 Leu Leu Val Val :.:.a Lcu Gly Ile Cly Leu Phe Me:t Flrg Arc3 Arg His GG~ GG5 G70 suesmu~~ sHE~r twin 2s~

WO 99/57981 PCI'NS99/10065 I lc Val Ar ~ Ly s :,rg Thr Leu Ar g Arc3 Le:u Lou Gln Giu Arg Glu Leu G'7 5 Val Glu Pro Leu ~nr Pro Ser Gly Gla Ala Pro As:. Gln Ala Leu Leu .. G95 7U0 Arc3 T_le Leu Lys G:.u Thr Glu Phe Lys Lys Ile Lys Vai Leu Giy Ser ,i5 720 Gly yla ?he Gly :':~r Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu Lys V41 Lye Ile ?wo V41 Ala Ile Lys Glu LW F:rg Giu Ala Thr Ser 74G ' . 745 750 ?ro Lys Ala as- Lys Glu Ile Leu Asp Glu T~la Tyr Val Met Aia S~:r Val asp Asn Pre is Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr VaI Gln i~e:: ~lc Thr Gln Leu Met ?ro Phc Gly Cys Leu Leu Asp Tyr Val Arg Gy;: is Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val G1~ ~'e Ala Lys Gly Met Asn 'fy_ Leu Glu Asp Arg Arg .. - 825 830 8?~
Leu Val His n.=.' ~~p Leu Ala Ala Arg A:i:: Val Leu Val Ly~ 'f hr Pro Gln His Val Lys .le Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys G:.;: ~yr His Ala Glu Gly Gly Lys Val Pro Ile LYs Trp Met Ala Leu G'_'.: Sor Ile Leu His Arg Ilc '1'y~r Thr His Gln Ser Asp Vai Trp Ser Ty_~ ply Val Thr Val 'frp Glu Li:u Met Thr Phe Gly Ser 9C~.
Lys Pro Tyr Fs~ ~ly -le Pro Ala Sir Glu Ile .'per Ser Ile Leu Glu SUBSTITUTE SHEET (RUL.E 26) Ly:; G:y Giu r.r~~ Lcu Pro Gln Pro Pro Iie Cy:: 't'hr lie App Val Tyr 9~0 935 940 iret ;~c btc;t Vai Lys Cy~ Trp MeL Ilc: :lip :?ia rl~p S~: Ary Pro Lys )~t ~ ~.'i0 ~)!~!; 9G0 Phe hrg Giu Leu Ile Ile Glu Phe Ser Ly:: A:i:t .~'~la ::=g Asp Pro Gln Arg iyr Leu Val le Gln Gly Asp Glu ::r c,~ M4i. l;is Lau Pro Ser Pro ~'hr rs? Ser Asn ?he Tyr Ary Ala Leu :~ic~ flsp Glu G.u Asp Met Asp 995 1000 lOCS
:;sp .',: Vai ?.sp .~'..a Asp Giu :yr Le:u :.;:c L'ro G.in G1:: Gly Phe L'he iG:~ 1015 1020 Ser Ser Pro Ser ~_':.r Ser Ary Thr Pr o Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro Iie Lys Glu Asp Ser Phe Lcu Gln Ary Tyr Ser Ser Asp lOGO lOGS 1070 Pro T: Giy Aia Leu Thr Glu Asp Ser T_le iap :asp ~r Phe Leu Pro ;075 lOBO iOVS
Val P~c Glu T:~r ~ie Asr. Gln Ser Val Pro Lys :arc _ro Ala Gly Ser Val Gin Asn Pro Val Tyr !-lis Asn Gln Pro Lcu tan ?ro Ala Pro Ser Ary :;sp ?ro His ".'yr Gln Asp L'ro I~li:: Si:r Thr t~la V4= Gly Asn Pro 1:25 1130 1135 Glu Ty: Leu As~ '"'.hr Val Gln Pro Thr Cy~ Val t~.sn Scr Thr Phe Asp Ser Pro .~':la His "'~? Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp =155 1160 liG:i Asn Pr o tap ~'yr Gin Gln Asp Phe Phe Pr J Lys Glu hia Lys Pro Asn 1:"; 1175 110 SUBS'TtTtJTE SHEET (RULE 2B) Gly Ile Pipe Lys Gly Scr Thr tlla Glu Asn Ala Clu ':fir Leu Arch Val ~~la :~~o Gln Scr Scr Glu ?hc Ile G~y Ala ;~05 i210 SU88TITUTE SHEET (RULE 2B)

Claims (20)

-13-

1. A method for active vaccination against autologous cells expressing transmembrane proteins comprising administering to a patient a vaccine composition comprising at least an immunogenic portion of the extracellular domain of the transmembrane protein, or a xenogeneic homolog thereof, coupled to or administered with an carrier protein effective to break tolerance to the transmembrane protein and a pharmaceutically acceptable adjuvant.

2. The method of claim 1, wherein the transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, the CD19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein.

3. The method of claim 1, wherein the transmembrane protein is CD20.

4. The method of claim 1, wherein the vaccine composition comprises a peptide having the sequence given by Seq. ID No 1 or 2.

5. The method claim 1, wherein the carrier protein is keyhole limpet hemocyanin.

6. The method of claim 5, wherein the transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, the CD19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein.

7. The method of claim 5, wherein the transmembrane protein is CD20.

8. The method of claim 7, wherein the vaccine composition comprises a peptide having the sequence given by Seq. ID No 1 or 2.

9. A method for active vaccination against B cells expressing CD20 comprising administering to a patient a vaccine composition comprising at least an immunogenic portion of the extracellular domain of CD20, or a xenogeneic homolog thereof, coupled to or administered with an carrier protein effective to break tolerance to the transmembrane protein and a pharmaceutically acceptable adjuvant.

10. The method claim 9, wherein the carrier protein is keyhole limpet hemocyanin.

11. The method of claim 9, wherein the vaccine composition comprises a peptide having the sequence given by Seq. ID No 1 or 2.

12. A method for treatment of B cell non-Hodgkin's lymphoma, comprising administering to a patient suffering from B cell non-Hodgkin's lymphoma a vaccine composition comprising at least an immunogenic portion of the extracellular domain of CD20, or a xenogeneic homolog thereof, coupled to or administered with an carrier protein effective to break tolerance to the transmembrane protein and a pharmaceutically acceptable adjuvant.

13. A vaccine composition comprising at least an immunogenic portion of the extracellular domain of the transmembrane protein, or a xenogeneic homolog thereof, coupled to or administered with an carrier protein effective to break tolerance to the transmembrane protein and a pharmaceutically acceptable adjuvant.

14. The composition of claim 13, wherein the transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, the CD19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein.

15. The composition of claim 13, wherein the transmembrane protein is CD20.

16. The composition of claim 15, wherein the vaccine composition comprises a peptide having the sequence given by Seq. ID No 1 or 2.

17. The composition of claim 13, wherein the carrier protein is keyhole limpet hemocyanin.

18. The composition of claim 17, wherein the transmembrane protein is selected from the group consisting of CD20, Her2-neu, VEGF receptor, epidermal growth factor receptor, the CD19 molecule, interleukin-2-receptor, interleukin-4-receptor, and the P-glycoprotein.

19. The composition of claim 17, wherein the transmembrane protein is CD20.

20. The composition of claim 19, wherein the vaccine composition comprises a peptide having the sequence given by Seq. ID No 1 or 2.