This invention provides for methods of immunizing mammals against tumors expressing a carcinoembryonic antigen using a carcinoembryonic immunogen modified to lack cellular immunosuppressive activity or CD1d binding. The invention further provides for use of a carcinoembryonic immunogen having a region recognized by antibody B9 or L12 modified to inactivate cellular immunosuppressive activity. Methods are also provided for modifying the immunogen by deleting, altering, mutating or truncating the immunosuppressive region. The invention also provides for enhancing cellular immunogenicity of an orally delivered immunogen by co-administration of an agent capable of inhibiting the immunosuppressive activity of carcinoembryonic antigen. Further methods are provided for creating a patient having a carcinoembryonic-antigen-family-member-expressing tumor by immunizing the patient with the carcinoembryonic-antigen-family-member lacking the immunosuppressive region. Pharmaceutical compositions for eliciting an effective immune response to the carcinoembryonic antigen are also provided.

Description of the Invention

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to a method for effectively immunizing an animal against a carcinoembryonic-antigen-family-member-expressing tumor by at least immunizing the animal using an immunogen consisting of a carcinoembryonic antigen family member lacking cellular immunosuppressive activity. The carcinoembryonic antigen family member lacking cellular immunosuppressive activity may be a carcinoembryonic antigen (CEA) family member which has an alteration or deletion of a region that is recognized by monoclonal antibody B9, or it may be GYSWYKGERVDGNRQII (SEQ ID NO:1), WYKGERV (SEQ ID NO:2) or YKGERVD (SEQ ID NO:3). The lacking of the cellular immunosuppressive region or activity may be achieved by altering, deleting, replacing, or otherwise modifying one or more amino acids in the cellular immunosuppressive region of the CEA family member molecule, such that the altered protein lacks the ability to suppress an immune response or induce suppressor T cells capable of suppressing an immune response. The suppression may be suppression of an antibody response, the suppression of a cytotoxic cellular response, or suppression of both responses. In a preferred embodiment, the carcinoembryonic antigen family member is carcinoembryonic antigen. The animal is preferably a mammal, most preferably a human.

The invention is also directed to compositions and pharmaceutical compositions comprising one or more CEA family members with an altered cellular immunosuppressive region such that the composition when used as a vaccine or immunogen is capable of eliciting an effective humoral and/or cellular immune response against the CEA family member, without the immunosuppressive activity contributed by the cellular immunosuppressive region of the CEA family member. Alterations of the region are exemplified above, but are not so limiting. Preferably, the CEA family member is carcinoembryonic antigen. The composition may further comprise a pharmaceutically-acceptable carrier, excipient, or diluent.

In a further aspect, the invention is directed to a method for effectively immunizing an animal against a tumor that is expressing a carcinoembryonic antigen family member by at least immunizing the animal with a carcinoembryonic antigen family member together with an agent capable of inhibiting a cellular immunosuppressive region of the carcinoembryonic antigen family member. The agent may be, for example, an inhibitor of the engagement of CD8 with a cellular immunosuppressive region of the carcinoembryonic antigen family member, such as a monoclonal antibody such as B9 or L12, or a CD8 peptide or CD8 fusion peptide capable of binding to the immunosuppressive epitope on a CEA family member. It also may be an inhibitor of the engagement of CD1d with a region of the carcinoembryonic antigen family member. Non-limiting examples of a CD8 peptide or fusion peptide include a CD8-Fc fusion peptide or a CD8 peptide comprising an epitope recognized by any one of antibodies OKT8B, OKT8E, OKT8F or OKT8I1. The monoclonal antibodies are preferably humanized monoclonal antibodies. Preferably the animal is a mammal, most preferably a human.

In still yet a further aspect, the invention is also directed to a method for enhancing the immunogenicity of an orally-delivered immunogen in an animal by at least co-administering the orally-delivered immunogen with an agent capable of inhibiting the cellular immunosuppressive activity of a carcinoembryonic antigen family member. In one embodiment, the agent is a monoclonal antibody capable of disrupting the engagement of a carcinoembryonic antigen family member with CD8 or CD1d, such as but not limited to monoclonal antibodies B9 or L12, or a CD8 peptide, CD8 fusion peptide, CD1d peptide or CD1d fusion peptide capable of binding to the immunosuppressive epitope on a CEA family member, or a CD1d peptide or CD1d fusion peptide capable of inhibiting the interaction of a CEA family member with CD1d. Non-limiting examples of such CD8 peptides and CD8 fusion peptides include a CD8-Fc fusion peptide or a CD8 peptide comprising an epitope recognized by any one of antibodies OKT8B, OKT8E, OKT8F or OKT8I1. A humanized monoclonal antibody is preferred. The enhanced immunogenicity may be a humoral, cellular, or both responses.

In yet still a further aspect of the invention, a method is provided for suppressing a humoral or cellular response to an antigen by providing at the site of the antigen a carcinoembryonic antigen, a carcinoembryonic family member, or a fragment, insertion, or fusion polypeptide of CEA or a CEA family member comprising the cellular immunosuppressive region, such that any potential immune response elicited by the antigen may be suppressed by the presence of the activity of the cellular immunosuppressive region of CEA or a CEA family member. Such a region is, for example, the region of CEA depicted in SEQ ID NO:1, or is a region that is recognized by monoclonal antibodies B9 or L12. In a further embodiment of this aspect of the invention, a protein comprising the cellular immunosuppressive region of a CEA family member may be introduced by genetic means into a site within the body, such as a cellular mass or tissue, such that expression and secretion of the protein comprising the cellular immunosuppressive region abrogates an immune response. This aspect of the invention is preferably applied to autoimmune diseases, such as but not limited to inflammatory bowel disease, ulcerative colitis, Crohn's disease, lupus, psoriasis, as examples of diseases in which an inappropriate immune response to an endogenous or exogenous antigen is pathogenetic.

It is thus an object of the invention to provide a method for effectively immunizing an animal against a carcinoembryonic-antigen-family-member-expressing tumor by immunizing the animal using an immunogen consisting of the carcinoembryonic antigen family member lacking an active cellular immunosuppressive region or CD1d binding. The active cellular immunosuppressive region includes a monoclonal antibody B9- or L12-recognizing region.

In a preferred embodiment, the active cellular immunosuppressive region is SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The aforementioned immunogen may have a deletion, alteration; mutation, or truncation of the active cellular immunosuppressive region which abrogates its cellular immunosuppressive activity. The cellular immunosuppressive activity may be directed to a humoral, or cellular response or both.

The carcinoembryonic antigen family member preferably is carcinoembryonic antigen.

It is another object of the invention to provide a method for effectively immunizing an animal against a tumor expressing a carcinoembryonic antigen family member by at least immunizing the animal with the carcinoembryonic antigen family member together with an agent capable of inhibiting a cellular immunosuppressive region of the carcinoembryonic antigen family member. The agent may be a ligand of the cellular immunosuppressive region, such as but not limited to a monoclonal antibody such as B9 or L12. The agent may be an inhibitor of the engagement of CD8 with a cellular immunosuppressive region of a carcinoembryonic antigen family member, or of the engagement of CD1d with a cellular immunosuppressive region of a carcinoembryonic antigen family member. Preferably, the carcinoembryonic antigen family member is carcinoembryonic antigen (CEA), and the mammal a human.

It is a further object of the invention to provide a method for enhancing cellular immunogenicity of an orally-delivered immunogen in an animal by co-administering with the orally-delivered immunogen an agent capable of inhibiting the cellular immunosuppressive activity of a carcinoembryonic antigen family member. The agent may be a ligand of the carcinoembryonic antigen family member capable of disrupting the engagement of the carcinoembryonic antigen family member with CD8 or CD1d, such as but not limited to a monoclonal antibody, such as B9 or L12. The agent is an inhibitor of the engagement of CD8 of CD1d with a cellular immunosuppressive region of the carcinoembryonic antigen family member. Preferably, the carcinoembryonic antigen family member is carcinoembryonic antigen, but it is not so limiting.

It is another object of the invention to provide a method for treating a patient having a carcinoembryonic-antigen-family-member-expressing tumor by at least immunizing the patient with an immunogen consisting of the carcinoembryonic antigen family member lacking a cellular immunosuppressive region. The CEA family member may be lacking a B9/L12 epitope having an alteration, deletion, mutation, or other change which abrogates the cellular immunosuppressive activity of the region.

It is yet another object of the invention to provide a method for suppressing a humoral or cellular immune response in a mammal to an antigen by at least administering to a site of the humoral or cellular response in the mammal an agent comprising a cellular immunosuppressive region of a carcinoembryonic antigen family member. The agent comprising a cellular immunosuppressive region of a carcinoembryonic antigen family member may be carcinoembryonic antigen, a carcinoembryonic antigen family member, or a fragment or fusion polypeptide of either of the foregoing comprising a cellular immunosuppressive region. The administering may be by introducing a vector capable of inducing expression of said agent in cells of said mammal. Preferably, the mammal is a human, and the carcinoembryonic antigen family member is carcinoembryonic antigen. This method is useful for the treatment of chronic inflammatory and autoimmune disorders.

It is still yet another object of the invention to provide pharmaceutical compositions for eliciting an effective immune response to a carcinoembryonic antigen family member comprising an immunogen consisting of the carcinoembryonic antigen family member lacking an active cellular immunosuppressive region. The cellular immunosuppressive region may be a monoclonal antibody B9- or L12-recognizing region, such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The lacking may be achieved by a deletion, alteration, mutation, or truncation of the active cellular immunosuppressive region. Preferably, the carcinoembryonic antigen family member is carcinoembryonic antigen.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors, in further studies of the intestinal epithelial cell glycoprotein gp180, have identified it as the oncofetal antigen known as carcinoembryonic antigen, or CEA. The attribution of the characteristics of gp180 to CEA, and the recognition that CEA is naturally expressed on intestinal epithelial cells, offers several new modalities for both improvements of mucosal vaccination in general, as well as a means for improving the immunogenicity of CEA, a popular yet seemingly recalcitrant target for immunotherapy for cancers that overexpress CEA. Moreover, as other CEA family members share common aspects with gp180, now recognized to be CEA, immunotherapetic approaches using other CEA family members, such as cancer immunotherapy, may also be enhanced.

In the aspect of the invention directed to enhancing the immune response specifically to CEA and its family members, the invention herein provides an explanation for the poor immunogenicity elicited by CEA in various cancer immunotherapy trials that have been performed to date, and offers a means for rectifying the poor immunogenicity. The inventors herein have identified an epitope of CEA extending from amino acids 30 to 46, GYSWYKGERVDGNRQII (SEQ ID NO:1), against which monoclonal antibodies raised thereto block the cellular immunosuppressive activity of CEA. Specifically, monoclonal antibodies B9 and L12, which recognize the epitopes WYKGERV (SEQ ID NO:2) and YKGERVD (SEQ ID NO:3), respectively, when included in an IEC:T cell culture, block the cellular immunosuppressive activity of CEA. Thus, elimination of the aforementioned epitope in vaccines or other immunogens against which an immune response to CEA is desired will avoid the cellular immunosuppressive activity of CEA and allow an effective anti-CEA cellular response to be elicited. Such an effective CEA immunogen may be achieved using fragments of CEA exclusive of the aforementioned cellular immunosuppressive region, or a fusion polypeptide which deletes the cellular immunosuppressive region from the CEA molecule.

The cellular immunosuppressive region of CEA or of a CEA family member is defined herein as a region: 1) that binds to CD8 and alters signaling such that cytotoxic T cells are not generated and 2) that activates regulatory or suppressor T cells which mediate immunosuppression. The latter activity is mediated by the interaction between a CEA family member and the class 1b molecule CD1d. Either or both of these activities may be suppressed, or enhanced, by the methods and agents of the invention.

The aforementioned strategies in achieving an effective cellular immune response against CEA, for the treatment of CEA-expressing cancers, may also be translated to other CEA family members, such as NCA, CGM-2, CGM6 and BGP, or, using newly-adopted nomenclature, CEACAM6, CEACAM7, CEACAM8 and CEACAM1, respectively; or, to other molecules expressing the B9/L12 epitopes described herein, for the treatment of cancers in which these CEA family member proteins are expressed and are thus targets for cancer immunotherapy.

In a further embodiment of the invention, agents and methods are provided for the treatment of CEA-expressing tumors beyond the methods described above, in which a more effective CEA vaccine is provided. The results presented here provide an explanation for the immunosuppression associated with these tumors. Moreover, increased expression of CEA in conjunction with any other tumor antigen explains the failure of the immune system to identify the appearance of such other tumor antigens. Thus, upon identification of the increased expression of CEA in any dysproliferative disease, a cause for general cellular immunosuppression is identified, and therapies to enhance a cellular response in the presence of the cellular immunosuppressive CEA may be initiated, as described herein, to elicit an effective cellular response. As will be seen below, gp180/CEA does not inhibit the effector function of cytotoxic T lymphocytes, so that if the therapies described herein successfully elicit a cellular immune response against the target antigen, be it CEA or another antigen, the effector cells will carry out their cytolytic activity against cells expressing the antigen.

As alluded to above, the invention extends beyond therapeutic modalities to enhance the immunogenicity of CEA and avoid a cellular immunosuppressive response. The identification of gp180 as CEA, in combination with the recognition that intestinal epithelial cells are antigen presenting cells, that CEA engages CD8 resulting in suppression of a cytotoxic T cell response, and that CEA activates a T suppressor cell response by association of gp180/CEA with CD1d as described in Campbell et al., 1999, J. Biol. Chem. 274:26259-26265, incorporated herein by reference in its entirety, provides a new means for enhancing the immunogenicity, particular of a cellular response, against any antigen, immunogen or vaccine that is presented by gp180/CEA-bearing or -expressing cells, such as intestinal epithelial cells. In a particular embodiment, such vaccines delivered mucosally, i.e., oral vaccines, may not elicit a desirably robust cellular immune response thereto for the reasons mentioned above and the invention is directed to oral vaccination in conjunction with a means for blocking the cellular immunosuppressive effects of gp180/CEA. While Applicants are not bound to theory, the finding of the immunosuppressive effects of gp180/CEA on CD8+ T cells and that CD1d restricted T cells inhibit other immune responses, the attendant inhibition of a cytotoxic T-cell response, and/or activation of T-suppressor cells, and their heretofore unknown and now surprising correlation with CEA, may be advantageously used in avoiding such immunosuppressive activities by the means described herein. Such means, as mentioned above, include co-administration of an agent which blocks the cellular immunosuppressive region of CEA, antibodies which bind to and block the cellular immunosuppressive activity of CEA, and other agents which block signalling resulting from the engagement of CD8 or CD1d with the aforementioned region of CEA. These are merely non-limiting examples of methods and agents which may be used to abrogate oral tolerance during desired immunization protocols.

Furthermore, in certain instances, immunosuppression is desirable, such that the cellular immunosuppressive region of a CEA family member is a useful activity. For example, in autoimmune diseases and other undesired or inappropriately robust immune responses, methods for abrogating a humoral and/or cellular immune response are desirable. For example, in various inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, as well as other localized and systemic autoimmune diseases including lupus and psoriasis, an undesired immune response exists, and flare-up of the disease has an immunological basis. In accordance with the present invention, use of a CEA family member which has an active cellular immunosuppressive region, or a truncation, fusion polypeptide, or other agent with the properties of the aforedescribed region of various CEA family members is useful for local or systemic therapy, disease dependent, for abrogating a humoral or cellular immune response. A congenital or acquired absence of CEA or CEA family member is believed to promote chronic inflammation; gene therapy to the affected cellular population or tissue provides a means for providing or increasing the production of CEA or CEA family member and the attendant and desirable, in this instance, suppression of a humoral and/or cellular response.

The preferred carcinoembryonic antigen family member immunogens for use in a vaccine for eliciting a cellular response to the carcinoembryonic antigen family member are modified to lack an active region of the carcinoembryonic antigen family member molecule wherein CD8 or CD1d engagement occurs. In the case of carcinoembryonic antigen, the area is shown generally in SEQ ID NO:1, and more specifically as SEQ ID NO:2, the epitope recognized by monoclonal antibody B9. Thus, a preferred CEA immunogen is lacking SEQ ID NO:1 or SEQ ID NO:2. The lacking of the region may be achieved by partial or total deletion, alteration of a critical amino acid which confers activity, substitution with another sequence to preserve the desired immunogenicity of the remainder of the molecule, formation of a fusion polypeptide or truncated molecule, all of which abrogate the cellular immunosuppressive activity of the CEA family member. A skilled artisan, based on understanding the present invention and the immunological activity conferred by the region of CEA identified herein, will readily design molecules without the activity. Corresponding deletions of related carcinoembryonic antigen family members are also embraced herein for use as immunogens or compositions in vaccines for the aforementioned uses.

In the instance where the cellular immunosuppressive region is desired, agents such as the immunosuppressive peptide portion of the CEA family member, or fusion polypeptides, may be provided which offer the activity of decreasing the immune response.

The foregoing modified carcinoembryonic antigen family member for use as a vaccine may be administered to an animal patient, preferably a mammalian patient, and most preferably a human patient, by routine methods known to elicit a cellular response for immunogens that normally elicit a cellular response. A preferred but non-limiting route is oral, mucosal, intradermal, intramuscular or subcutaneous. The immunogens of the invention lacking the activity of the cellular immunosuppressive region elicit a therapeutic or protective cellular response and thus are capable of an antibody or cytotoxic activity against cells expressing the carcinoembryonic antigen family member.

In a further aspect, the cellular response of an orally- or mucosally-delivered immunogen other than a carcinoembryonic antigen family member may be enhanced by blocking the cellular immunosuppressive activity of a carcinoembryonic antigen family member, preferably carcinoembryonic antigen, during antigen presentation. Agents which disrupt the engagement of the T cell CD8 molecule and the cellular immunosuppressive region of the carcinoembryonic antigen family member, or block the association of the carcinoembryonic antigen family member with CD1d are co-administered to block the effect. The co-administered agent may be administered by the same route or a different route than the immunogen, to provide effective blockage of the cellular immunosuppressive effect. For example, a small-molecule antagonist of the carcinoembryonic antigen family member-CD8 interaction may be included in the oral vaccine, or may be administered parenterally to the individual. In another embodiment, an agent capable of disrupting the signalling events following carcinoembryonic antigen family member-CD8 engagement may be administered.

As noted above, carcinoembryonic antigen family member deletion mutants useful for eliciting a cellular immune response are embraced herein. Such deletion mutants, or fragments lacking the cellular immunosuppressive region, may be prepared by routine methods and then screened for successful elimination of cellular immunosuppression in an in-vivo assay. These assays for effective induction of a CTL response are routine and readily undertaken by one skilled in the art.

Candidate patients or individuals for the therapeutic and protective therapies of the invention include animals, preferably mammals including livestock and domestic animals, and most preferably humans. While the expression of various CEA family members among non-human mammalian species has been little studied, the teachings herein are applicable notwithstanding the member(s) of the family expressed in a particular species. One target patient population for the methods herein is that with a carcinoembryonic antigen family member-expressing tumor in which control or eradication of the tumor is desired. Traditionally, carcinoembryonic antigen family member immunogens have been unsuccessful at mounting an effective cellular response, now understood based on the present invention to be the presence of a cellular immunosuppressive epitope amongst the carcinoembryonic antigen family member epitopes. The epitope appears responsible for both suppression of CD8 signalling and the induction of cytotoxic T cells, as well as activation of T cells with suppressor activity. In another embodiment, the cellular immune response to another antigen, such as an infectious disease immunogen or other cancer immunogen, particularly one delivered orally and presented by mucosal cells, may be enhanced by blocking the cellular immunosuppressive activity of the carcinoembryonic antigen family member, particularly carcinoembryonic antigen, now known to be present in intestinal (epithelial) cells and responsible based herein for reducing the effectiveness of immunogens presented thereby. Moreover, the CD8 T-suppressor cells generated by CEA are not target restricted. Vaccines comprising immunogens which enter the immune system through the intestinal epithelial cell are merely exemplary of such vaccines for which an enhanced cellular immune response is achievable by the methods herein.

The invention is also directed to pharmaceutical compositions of the aforementioned CEA family member molecules lacking cellular immunosuppressive activity, and to their use, for example, as an effective immunogen for a CEA family member-expressing tumor. Examples of such altered CEA family member molecules include CEA with an N domain deletion, or a K35A substitution in the N domain, or a deletion of GYSWYK or NRQII. These are described in Taheri, et al., 2000, J. Biol. Chem. 275:26935-43, which is incorporated herein by reference in its entirety. These molecules may be formulated with appropriate pharmaceutically-effective carriers, excipients or diluent for local or systemic administration, as appropriate for the prophylaxis or treatment of a particular disease or condition.

As noted above, previous studies have suggested that normal intestinal epithelial cells can act as nonprofessional antigen presenting cells, selectively activating CD8.sup.+ suppressor T cells. As will be shown in the examples below, an epithelial cell surface glycoprotein, initially called gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical to the activation of these regulatory T cells. Purification and sequence analysis of mAb B9 reactive material revealed sequence homology with carcinoembryonic antigen (CEA). Transfection of CEA cDNA into CHO cells, 293T cells and F01 cells (melanoma line) conferred mAb B9 reactivity to these transfectants. Properties previously attributed to gp180 such as CD8.alpha. binding and activation of CD8 associated p561ck could be reproduced using purified CEA from these transfectants. The mAb B9 epitope was determined to be in the N domain characterized by the sequence GYSWYKGERVDGNRQII (SEQ ID NO:1). This sequence is found with some variation in other CEA subfamily members as well. These data suggest that CEA may be an important immunoregulatory molecule expressed in the normal intestine and that increased expression in malignancy may explain the immunosuppression associated with these tumors.
 

Claim 1 of 10 Claims

1. A method for immunizing an animal against a tumor expressing a carcinoembryonic antigen comprising immunizing said animal using an immunogen consisting of said carcinoembryonic antigen modified to lack cellular immunosuppressive activity or CD1d binding, wherein said carcinoembryonic antigen comprises an N domain deletion, a K35A substitution in the N domain, or a deletion of GYSWYK (SEQ ID NO: 10) or NRQII (SEQ ID NO: 11).

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