How tumours escape mass destruction.

It is now well established that the immune system can control neoplastic development and growth in a process termed immunosurveillance. A link between host immunosurveillance and neoplastic progression is revealed in cases where the immune response becomes compromised due to genetic or other pathological conditions, resulting in a substantially increased incidence and rate of spontaneous tumour formation in both preclinical animal models and patients. It has also been demonstrated in tumour-bearing hosts that the tumorigenic process itself can promote a state of immunosuppression that, in turn, facilitates neoplastic progression. The ability of neoplastic populations to induce a hostile microenvironment through both cell contact-dependent and -independent immunosuppressive networks is a significant barrier to effective cell-mediated immunity and immunotherapy. Thus, a competent immune system is integral for the control of neoplastic disease, and dissecting the plethora of tumour escape mechanisms that disrupt this essential host defense capability is integral for the development of effective immunotherapeutic paradigms.

Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8(+) CTL response in vivo, as well as its effectiveness against an "extensive" tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2L(d)-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (> or =1 x 10(6)) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (> 150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.

Positive and negative consequences of soluble Fas ligand produced by an antigen-specific CD4(+) T cell response in human carcinoma immune interactions.

The influence of a human CD4(+) T cell response in anti-carcinoma immune reactions remains largely uncharacterized. Here, we made use of a major histocompatibility complex (MHC) class-II-restricted, anti-ras oncogene-specific CD4(+) T cell line produced previously in vivo from a patient with metastatic carcinoma in a peptide-based phase I trial. Using this patient-derived T cell line as a potentially relevant cell type, we examined the consequences of the anti-carcinoma CD4(+) T cell response, with emphasis on specific lymphokines potentially important for the regulation of Fas/Fas ligand (FasL) interactions. Antigen (Ag)-specific CD4(+) T cells produced substantial amounts of IFN-gamma following recognition of MHC class-II-matched Ag-presenting cells expressing the cognate peptide. The IFN-gamma promoted significant upregulation of Fas on the surface of colon carcinoma cells and sensitized these targets to Fas-mediated apoptosis and Ag-specific CD8(+) cytotoxic T lymphocyte (CTL)-mediated lysis involving a Fas-based effector mechanism. Moreover, Ag-stimulated CD4(+) T cells secreted soluble FasL (sFasL), which induced the death of TNF-resistant/refractory colon, breast, and ovarian carcinoma cells. Interestingly, although CD4(+)-derived sFasL expressed cytotoxic activity, the recovery of carcinoma cells which resisted Fas-mediated lysis displayed enhanced metastatic ability in vivo, compared with the unselected parental population, in an athymic mouse model. Thus, a tumor-specific CD4(+) T cell response may have both positive and negative consequences in human carcinoma via the production of proinflammatory cytokines such as IFN-gamma and/or sFasL that may (1) improve or facilitate CTL-target engagement, contact-independent effector mechanisms, and the overall lytic outcome and (2) potentially select for Fas-resistant tumor cells that escape immune destruction, which may thus impact the metastatic process.

Characterization of CD8+ cytotoxic T lymphocyte/tumor cell interactions reflecting recognition of an endogenously expressed murine wild-type p53 determinant.

p53 mutations are frequently found in human cancers and are often associated with the overexpression of wild-type (WT) protein or peptide sequences, supporting the notion that WT p53 epitopes may serve as potential targets for tumor immunotherapy. We have developed a cytotoxic T lymphocyte (CTL)/p53 tumor-associated antigen (TAA) model, based on immune recognition of a WT p53 determinant. WT p53-peptide-specific, major histocompatibility complex (MHC) classI-restricted CTL were produced from immunocompetent C57BL/6 (H-2b) mice after immunization with a previously defined WT p53 peptide (p53(232-240)) Epitope-specific CTL were then employed to identify syngeneic tumor cell populations expressing that antigenic determinant. Two syngeneic tumor cell lines, MC38 colon carcinoma and MC57G fibrosarcoma, were demonstrated to express the endogenous WT p53(232-240) determinant naturally, as defined by CD8 + CTL recognition. Cold-target inhibition assays confirmed that CTL-mediated lysis was due to immune recognition of the p53(232-240) peptide epitope. The p53(232-240)-specific CTL line did not lyse syngeneic normal cells (i.e., mitogen-activated splenocytes) in the absence of exogenous peptide, suggesting that the WT-p53-specific CTL could distinguish between tumor cells expressing self-TAA and normal host cells. We have demonstrated, for the first time, that the adoptive transfer of WT-p53-specific CTL to mice with established pulmonary metastasis resulted in antitumor activity in vivo. The ability to generate MHC-class-I-restricted CD8- CTL lines specific for a non-mutated p53 determinant from normal, immunocompetent mice, which display antitumor activity both in vitro and in vivo (by adoptive transfer), may have implications for the immunotherapy of certain p53-expressing malignancies.

Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes.

Certain anti-neoplastic agents at subtoxic doses may exert immunomodulatory effects, which alter the expression of specific tumor cell surface molecules. We reasoned that potential increases in tumor cell surface markers, such as those important for facilitating effector-target contact, as well as triggering cell death pathways, might then improve antigen (Ag)-specific T-cell-mediated tumor cytolysis. Here, in a human colon carcinoma cell model in vitro, we examined whether the anti-neoplastic agents 5-fluorouracil (5-FU), CPT-11 or cisplatin (CDDP) could upregulate the expression of specific tumor cell surface markers, which may then enhance productive lytic interactions between CD8+ CTL and Ag-bearing tumor cells. Based on our earlier studies, IFN-gamma treatment was included as a control for sensitization to CTL-mediated lysis. Pretreatment of the SW480 primary colon carcinoma cell line with IFN-gamma, 5-FU, CPT-11 or CDDP enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis involving Fas-dependent and -independent mechanisms. In contrast, pretreatment of the SW620 metastatic isolate, derived from the same patient, with IFN-gamma, CPT-11 or CDDP, but not 5-FU, enhanced ICAM-1 expression, resulting in Ag-specific CTL-mediated lysis via Fas-independent mechanisms only. Flow cytometric-based assays were then developed to measure the effects of drug treatment on caspase signaling and apoptosis incurred by tumor targets after interaction with CTL. We found that the lytic enhancement caused by drug treatment of SW480 or SW620 targets was accompanied by an increase in caspase-3-like protease activity. A peptide-based caspase inhibitor abrogated CTL-mediated apoptosis, suggesting that "chemomodulation" involved regulation of the caspase pathway. These results revealed for the first time an important role for components of the caspase pathway, such as caspase-3-like proteases, in the sensitization of human colon carcinoma cells by anti-neoplastic agents to Ag-specific CTL. Thus, certain anti-neoplastic agents may display unique immunoregulatory properties that facilitate human colon carcinoma death by engaging the lytic capacity of Ag-specific CTL, which may have implications for chemoimmunotherapy strategies.

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization.

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ¿i.e., 4-16(Val12)¿. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ¿i.e., 4-12(Val12)¿, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ¿i.e., 5-17(Val12)¿ lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.

Influence of interferon gamma on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack.

The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon gamma (IFNgamma). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8+ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2+ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFN-gamma was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNgamma influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNgamma pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNgamma-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the alpha T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNgamma also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNgamma (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNgamma may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack.

Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL.

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-gamma treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-gamma pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-gamma and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential "escape" of Fas-resistant carcinoma cells.

Rational antigen modification as a strategy to upregulate or downregulate antigen recognition.

Recent and rapid advances in our understanding of the cellular and molecular mechanisms of antigen recognition by CD8(+) and CD4(+) T lymphocytes have led to the birth of possibilities for site-directed, rational modification of cognate antigenic determinants. This immunologic concept has vast biomedical implications for regulation of host immunity against the pathogenesis of diverse disease processes. The upregulation of antigen-specific T-cell responses by 'agonistic' peptides would be most desirable in response to invasive pathogenic challenges, such as infectious and neoplastic disease, while the downregulation of antigen-specific T-cell responses by 'antagonistic' peptides would be most efficacious during inappropriate pathologic consequences, such as autoimmunity. The capacity to experimentally manipulate intrinsic properties of cognate peptide ligands to appropriately alter the nature, course and potency of cellular immune interactions has important potential in both preventive and therapeutic clinical paradigms.

Strategies in the development of recombinant vaccines for colon cancer.

A new era involving the evaluation of recombinant vaccines for colon cancer has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include (I) the identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofetal antigens, and tissue lineage determinants; (2) the development of a variety of viral and bacterial vectors to deliver and present gene products; (3) the identification of numerous T-cell costimulatory molecules and the knowledge of their mode of action; (4) the cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules; and (5) a more sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation.

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.

A phase I vaccine trial with peptides reflecting ras oncogene mutations of solid tumors.

Mutations in the ras genes occur in 20% of all human cancers. These genes, in turn, produce mutated proteins that are unique to cancer cells, rendering them distinguishable from normal cells by the immune system. Thus, mutated Ras proteins may form potential targets for immune therapy. We conducted a phase I/pilot clinical trial in patients with advanced cancers to test the toxicity and the ability to induce an immune response by vaccination with 13-mer mutated Ras peptides reflecting codon 12 mutations. These peptides corresponded to each of the patient's own tumor Ras mutation. Patients were vaccinated monthly x3 subcutaneously with the specific Ras peptide along with Detox adjuvant (RiBi ImmunoChem Research, Inc., Hamilton, MT, U.S.A.) at one of five different peptide dose levels (100, 500, 1,000, 1,500, and 5,000 micrograms). Three out of 10 evaluable patients generated a mutant Ras specific CD4+ and/or CD8+ T-cell immune response. The CD8+ cytotoxic cells specific for Gly to Val mutation at codon 12 were capable of lysing an HLA-A2-matched tumor cell line carrying the corresponding mutant but not the wild-type ras gene. The treatment has been well tolerated with no evidence of serious acute or delayed systemic side effects on any of the five dose levels. We demonstrated that we can generate in cancer patients specific T-lymphocyte responses that detect single amino acid differences in Ras oncoproteins. Neither the immune responses nor the minor side effects seen were found to be dose dependent. This approach may provide a unique opportunity for generating a tumor-directed therapy. Also, in vitro stimulation of these cells with the corresponding peptide generated specific T-cell lines that could be used for adoptive immune therapy.

Identification of a human CD8+ T lymphocyte neo-epitope created by a ras codon 12 mutation which is restricted by the HLA-A2 allele.

Point mutations in the ras proto-oncogenes, notably at codon 12, are found in high frequency of human malignancies and, thus, may be appropriate targets for the induction of tumor-specific T cell responses in cancer immunotherapy. In this study, we examined the mutant ras protein sequence reflecting the substitution of Gly to Val at position 12 as a putative point-mutated determinant for potential induction of an HLA-A2-reactive, CD8+ cytotoxic T lymphocyte (CTL) response. We identified the ras 4-12(Val12) sequence as a minimal 9-mer peptide, which displayed specific binding to HLA-A2 by T2 bioassays. Peptide binding to HLA-A2 on T2 cells was weak and required coincubation with exogenous beta(2)-microglobulin to facilitate and enhance complex formation. In contrast, the wild-type ras 4-12(Gly12) peptide failed to bind to HLA-A2 even in the presence of beta(2)-microglobulin, consistent with the hypothesis that the point mutation creates a C-terminus anchor residue. A CD8+ CTL line against the ras 4-12(Val12) peptide was derived in vitro from a normal HLA-A2+ donor using a model culture system consisting of T2 cells as antigen presenting cells pulsed with exogenous mutant ras peptide and beta(2)-microglobulin plus cytokines (interleukin-2 and 12). Functional characterization of CD8+ CTL line revealed (1) peptide-specific and HLA-A2-restricted cytotoxicity against a panel of peptide-pulsed targets; (2) no specific lysis using the normal ras peptide sequence; (3) half-maximal lysis with exogenous peptide of approximately 0.3 microM; (4) lysis of HLA-A2+ B cell lines infected with a recombinant vaccinia virus construct encoding the point-mutated human K-ras gene; and (5) specific lysis of the HLA-A2+ SW480 colon carcinoma cell line expressing the naturally occurring K-ras Val12 mutation. Maximal lysis of SW480 cells occurred following interferon (IFN)-gamma pretreatment, which correlated with enhanced HLA-A2 and ICAM-1 (CD54) expression. Specificity of lysis was revealed by the absence of lysis against a HLA-A2+ melanoma cell line (+/- IFN-gamma), which lacked the mutant Val12 mutation, and the inability of an irrelevant CD8+ CTL line to lyse SW480 (+/- IFN-gamma) unless the appropriate exogenous peptide was added. These findings demonstrated that tumor cells may endogenously process and express mutant ras epitopes, such as the 4-12(Val12) sequence, albeit in limiting amounts that may be potentiated by IFN-gamma treatment. These data support the biological relevance of this sequence and, thus, may have important implications for the generation of ras oncogene-specific CTL responses in clinical situations.

Development of a murine mutant Ras CD8+ CTL peptide epitope variant that possesses enhanced MHC class I binding and immunogenic properties.

We recently identified a murine mutant Ras p21 CD8+ CTL epitope reflecting residues 4 to 12, containing the mutation of Gly to Val at codon 12, that bound weakly to H-2Kd in vitro and generated a weak primary CTL response in immunized BALB/c mice. Here, we explored the hypothesis that specific modifications to the Ras4-12 peptide sequence can improve MHC binding, leading to enhanced immunogenicity without altering immune specificity. We synthesized Ras4-12 peptides in which Val at residue 12 was replaced with the more dominant H-2Kd C-terminus anchor residue Leu or Ile. In functional H-2Kd binding assays, Ras4-12(L12 or I12) peptide variants competed more effectively than the Ras4-12(V12) peptide. Ras4-12(L12 or I12) peptide variants enhanced both in vitro cytotoxicity and proliferation responses of anti-Ras4-12 CTL compared with the mutant Ras4-12(V12) peptide. Additionally, the Ras4-12(L12) peptide variant induced a quantitatively greater T cell response in vivo compared with that produced by Ras4-12(V12) as determined by IFN-gamma production. Mice immunized with Ras4-12(L12) peptide elicited CD8+ CTL activity specific for target cells presenting the Ras4-12(V12) epitope exogenously and endogenously. Moreover, both anti-Ras4-12(V12)-derived and anti-Ras4-12(L12)-derived CTL lines were similar insofar as their TCR usage and amino acid contact residues in the Ras4-12(V12) peptide. These experiments demonstrate that modifications can be introduced in tumor-specific peptide epitopes to enhance both in vitro and in vivo immunogenicity. The design of oncogene-specific peptide epitope variants as immunogens may accelerate the generation of anti-tumor T cell responses for cancer immunotherapy.

Cancer vaccine development.

A new era involving the evaluation of recombinant cancer vaccines has begun with the concurrent emergence of insights and technologies in the fields of molecular biology and immunology. These advances include: The identification and cloning of an array of genes associated with the neoplastic process, such as oncogenes, suppressor genes, genes encoding oncofoetal antigens and tissue-lineage determinants. The development of a variety of viral and bacterial vectors to deliver and present gene products. The identification of numerous T-cell costimulatory molecules and an understanding of their mode of action. The cloning and analysis of the modes of action of an array of cytokines and other immunomodulatory molecules. More sophisticated knowledge of the mode(s) of antigen presentation and T-cell activation. One current challenge in cancer therapy is the delineation of strategies toward the rational design and implementation of recombinant vaccines that will be of therapeutic benefit to cancer patients and/or members of groups at high risk for specific neoplasias. Numerous concepts are emerging in this regard. The study of immunologic intervention using laboratory animal models demonstrates that no one approach will prevail for all cancer types or, perhaps, for the various stages of the neoplastic process of a given tumour type. The immunological role(s) of CD8+, CD4+, natural killer and other cell types, as well as the roles of antibodies, must all be taken into consideration. This article reviews some of the strategies currently undergoing evaluation toward the development of recombinant vaccines for several carcinoma types.

Construction and characterization of a recombinant vaccinia virus expressing murine intercellular adhesion molecule-1: induction and potentiation of antitumor responses.

The intercellular adhesion molecule-1 (ICAM-1) has been associated with cellular migration into inflammatory sites and with facilitating interactions between lymphocytes and tumor targets in the pathway of cell-mediated cytotoxicity. More recently, ICAM-1 has become increasingly implicated in the costimulation of T cell functions, such as antigen-dependent T cell proliferation. Previous murine studies have shown that the introduction of the ICAM-1 gene into tumor cells using retroviral vectors led to enhanced antitumor responses. In this study, we report the construction, characterization, and immunological consequences of a recombinant vaccinia virus expressing murine ICAM-1. Vaccinia virus represents an attractive vector for the delivery of molecules such as ICAM-1 due to its wide host range, rapid infection, and functional expression of inserted gene products. The infection of tumor cells with this recombinant virus resulted in the expression of functional ICAM-1. Infected tumors provide accessory or secondary signals to lymphoblasts in vitro, resulting in enhanced cytokine production or alloreactive cytotoxic T lymphocyte (CTL) activity. In vivo, we demonstrated that weakly immunogenic syngeneic tumors, infected with and expressing rV-ICAM-1, were rejected by immunocompetent hosts. Furthermore, immunization with rV-ICAM-1-infected tumors resulted in the rejection of subsequent tumor challenge, providing evidence for recall response and immunological memory. These studies demonstrated the utility of a recombinant vaccinia virus to deliver and efficiently express ICAM-1 molecules on tumor cells for potential gene therapy and recombinant approaches to cancer immunotherapy.

Adoptive immunotherapy as an in vivo model to explore antitumor mechanisms induced by a recombinant anticancer vaccine.

We have described previously the construction, generation, and in vivo biologic consequences of a recombinant vaccinia virus containing the human CEA gene (rV-CEA) in an experimental murine colon carcinoma model. Immunization of C57BL/6 mice with rV-CEA led to antigen-specific inhibition of tumor growth in both prophylactic and therapeutic settings. Although such antitumor effects were correlated with the induction of CEA-specific T-cell responses, their exact contribution in the tumor rejection mechanism remained unclear. In this study, we examined the mechanism of action of rV-CEA, with emphasis on definition of the immune cells important for such antitumor effects. To that end, a cellular adoptive transfer model was established in vivo, which allowed specific functional analysis of donor-derived immune cells in naive, sublethally irradiated, tumor-bearing recipients. Splenocytes from rV-CEA-immunized donors expressed strong antitumor activity in such tumor-bearing recipients, whereas nonimmune donor cells did not. Depletion of immune T cells before cellular transfer abolished the antitumor response. Moreover, depletion of CD8+ T cells before transfer resulted in the loss of antitumor activity, despite the presence of CD4+ T cells. In contrast, antitumor activity was demonstrable with CD8-containing, CD4-depleted effectors, although it was not as effective as with both T-cell subpopulations combined. Finally, in beta 2-microglobulin/CD8+ T-cell-deficient mice, rV-CEA immunization exerted only partial antitumor protection, compared with the immune-competent controls. Overall, we demonstrated that (a) antitumor activity induced by rV-CEA was essentially mediated by CD8+ effectors; and (b) the combination of both CD8+ and CD4+ lymphocytes led to maximal antitumor therapeutic effects, suggesting an important helper or immunoregulatory contribution of the CD4+ subset. Thus, adoptive cellular transfer strategies may have implications for both the study of recombinant anticancer vaccines and the development of potential clinical applications for cancer immunotherapy.

Generation of stable CD4+ and CD8+ T cell lines from patients immunized with ras oncogene-derived peptides reflecting codon 12 mutations.

Previous studies have identified and characterized both murine in vivo and human in vitro T cell responses reflecting specific mutations in the ras proto-oncogenes at codon 12, 13, or 61. In an attempt to determine whether peptide epitopes reflecting point mutations in the ras oncogenes are immunogenic in humans for the production of CD4+ and/or CD8+ T cell responses, a phase I clinical trial was initiated in metastatic carcinoma patients whose primary tumors harbor mutations in the K-ras proto-oncogenes at codon 12. The peptides used here as immunogens, which were administered in Detox adjuvant, spanned the ras sequence 5-17 and reflected the amino acid substitution of glycine (Gly) at position 12 to aspartic acid (Asp), cysteine (Cys), or valine (Val). Three of eight evaluable patients have demonstrated peptide-specific cell-mediated immunity, as determined by the production of T cell lines resulting from the vaccination. First, an antigen (Ag)-specific, major histocompatibility complex (MHC) class II (DP)-restricted CD4+ T cell line was established in vitro from postvaccination lymphocytes of a non-small cell lung carcinoma patient whose primary tumor contained a Cys12 mutation when cultured on the immunizing peptide. Moreover, CD4+ proliferation was inducible against the corresponding mutant K-ras protein, suggesting productive T cell receptor recognition of exogenously processed Ag. Second, an Ag-specific, MHC class I (HLA-A2)-restricted CD8+ cytotoxic T lymphocyte (CTL) line was established in vitro from postvaccination lymphocytes of a colon carcinoma patient whose primary tumor contained an Asp12 mutation. To that end, a 10-mer peptide, nested within the 13-mer immunizing peptide, was identified [i.e., ras5-14(Asp12)], which was shown to bind to HLA-A2 and display specific functional capacity for expansion of the in vivo primed CD8+ CTL precursors. Third, both Ag-specific, MHC class II (DQ)-restricted CD4+ and MHC class I-restricted (HLA-A2) CD8+ T cell lines were generated from a single patient with duodenal carcinoma whose primary tumor contained a Val12 mutation when cultured on the immunizing 13-mer peptide or a nested 10-mer peptide [i.e., ras5-14(Val12)], respectively. Evidence is thus provided that vaccination with mutant ras oncogene peptides in adjuvant may induce specific anti-ras cellular immune responses, with no detectable cross-reactivity toward normal proto-ras sequences. Moreover, we have identified for the first time human HLA-A2-restricted, CD8+ CTL epitopes reflecting specific point mutations in the K-ras oncogenes at codon 12 which, in concert with the activation of the CD4+ T cell response, may have important implications for both active and passive immunotherapies in selected cancer patients.

Comparison of linear and branched peptide forms (MAPs) in the induction of T helper responses to point-mutated ras immunogens.

The utility of multiple antigenic peptides (MAPs) for the induction of antibody and cellular immune responses in animal models has been demonstrated for a variety of peptide epitopes involved in human disease. However, little is known about immune responses to MAPs constructed with antigenic tumor epitopes, nor has peptide specificity in branched forms been addressed. A potentially important advantage of the MAP system over linear peptide immunogens for clinical applications is elimination of the need for a protein carrier with its associated toxicity and immunogenicity. Here, we examined cellular immune responses following in vivo administration of MAPs incorporating a 13-mer T helper epitope from point-mutated ras p21 (ras V12) and compared the potency of the responses to that of the linear peptide. The Gly --> Val mutation in position 12, which is associated with a range of human carcinomas, represents a useful system for evaluating the specificity of the immune response. In initial studies with the point-mutated linear peptide epitope, optimal in vitro proliferation responses were obtained following sc administration of the peptide in a squalane-containing adjuvant formulation. Comparative immunization studies using point-mutated MAPs bearing two, four, or eight branches were administered either in saline or in adjuvant. These studies showed that adjuvant was required for the induction of cellular immune responses using both linear and all three forms of branched peptides. Moreover, there was no apparent advantage of using any of the MAPs vs linear peptide when equivalent mass amounts were administered, i.e., the intensity of the immune response was no greater using any of the branched structures compared to the linear form. Specificity of the in vivo responses for both the linear and the MAP immunogens was demonstrated by the higher stimulation indices observed in vitro in the presence of the mutant ras V12 vs the normal ras G12 linear peptide. No apparent cellular immune response to the MAP core structure itself was observed. However, a nonspecific response to the two-branched MAP2G12 structure was observed in some assays, the nature of which is unknown at this time. This work represents the first reported investigation of a cellular immune response using MAP immunogens incorporating a tumor-specific peptide epitope and demonstrates that linear peptides are as efficient as three different MAP structures in the generation of specific T cell responses.

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses.

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8+ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2Kd; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8+ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4+ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2Kd was demonstrated with the mutant ras4-12(V12) peptide, but not the normal ras4-12(G12) peptide, which specifically inhibited an H-2Kd-restricted, anti-nucleoprotein NP147-155 CTL response in a dose-dependent fashion. An anti-ras CD8+ T cell line was then established from immune splenocytes of BALB/c (H-2d) mice injected with ras4-12 (V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2Kd and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12(Gly-->Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras4-12(V12) peptide was immunogenic for the production of antigen-specific CD8+ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.