Lakshminarayanan, Vani

AbstractA fully synthetic MUC1‐based cancer vaccine was designed and chemically synthesized containing an endogenous helper T‐epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody‐dependent cell‐mediated cytotoxicity (ADCC). It also activated cytotoxic T‐lymphocytes. Collectively, the immunological data demonstrate engagement of helper T‐cells in immune activation. A synthetic methodology was developed for a penta‐glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1‐based cancer vaccines that elicited potent immune responses employed exogenous helper T‐epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T‐epitope will be more attractive, because it will activate cognate CD4+ T‐cells that will provide critical tumor‐specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.

Cancer vaccines have often failed to live up to their promise, although recent results with checkpoint inhibitors are reviving hopes that they will soon fulfill their promise. Although mutation-specific vaccines are under development, there is still high interest in an off-the-shelf vaccine to a ubiquitous antigen, such as MUC1, which is aberrantly expressed on most solid and many hematological tumors, including more than 90% of breast carcinomas. Clinical trials for MUC1 have shown variable success, likely because of immunological tolerance to a self-antigen and to poor immunogenicity of tandem repeat peptides. We hypothesized that MUC1 peptides could be optimized, relying on heteroclitic optimizations of potential anchor amino acids with and without tumor-specific glycosylation of the peptides. We have identified novel MUC1 class I peptides that bind to HLA-A*0201 molecules with significantly higher affinity and function than the native MUC1 peptides. These peptides elicited CTLs from normal donors, as well as breast cancer patients, which were highly effective in killing MUC1-expressing MCF-7 breast cancer cells. Each peptide elicited lytic responses in greater than 6/8 of normal individuals and 3/3 breast cancer patients. The CTLs generated against the glycosylated-anchor modified peptides cross reacted with the native MUC1 peptide, STAPPVHNV, suggesting these analog peptides may offer substantial improvement in the design of epitope-based vaccines.

A strategically designed sialyl-Tn building block was synthesized in a stereospecific manner for the rapid linear construction of a glycolipopeptide cancer vaccine.

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O -linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam 3 CysSK 4 , a peptide T helper epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.

Abstract MUC1 is an excellent target for immunotherapeutic strategies for cancers expressing Mucin 1 (breast, pancreas etc). MUC1 was recently selected by the NCI working group as the number 2 cancer vaccine target antigen, based on its immunogenicity, oncogenicity and wide-spread cell-surface expression. More than 90% of breast cancers over express MUC1, it stimulates both humoral and cellular immunity, its normal apical distribution is lost in cancer cells and aberrant glycosylation exposes peptide epitopes and novel carbohydrate antigens. Clinical trials using unglycosylated MUC1 epitopes have resulted in MUC1 specific cytotoxic T lymphocytes (CTLs) with limited effects on clinical response. Patients with breast tumors have exhibited spontaneous immune responses to MUC1 with the presence of T cells and antibodies specific for glycosylated MUC1. Because these natural immune responses are often not efficient in eradicating the tumor, it is necessary to specifically modulate these immune responses. Hence it is possible that better immunogenicity would be obtained using peptides more representative of the novel (glycosylated) forms of MUC1 as seen in cancer to which individuals may be less tolerant. The presentation of antigenic peptides by MHC class I molecules is crucial for the development of vaccines. MUC1 peptides from the VNTR region, which has five potential glycosylation sites, can be presented by a variety of class I molecules and can generate MUC1 specific CTLs. But these epitopes lack definite anchors and bind with low affinity compared to high affinity peptides. Hence, we developed several peptides from the MUC1 H-2Kb peptide (SAPDTRPAP) epitope with all possible modifications to improve their MHC and TCR binding efficiency. Preclinical studies were performed using the modified H-2Kb-restricted peptides along with the DC stimulants (CpG-ODN and GM-CSF) and foreign helper epitope (hepatitis B virus optimized I-Ab T helper peptide) in incomplete Freund's adjuvant in C57BL/6 mice transgenic for human MUC1. Peptides that were glycosylated at threonine and had methionine or leucine as the terminal amino acid induced a strong CTL response and were able to overcome tolerance in MUC1.Tg.mice. In the breast cancer model with the MUC1- expressing MMT cell line, the onset of tumor was delayed with peptide immunization and there was significant reduction in the tumor weight. Increased lytic activity of CD8+ CTLs and infiltration of T lymphocytes to the tumor site were observed with modified peptides as compared to the effect of MHC class I Kb and Db restricted native MUC1 peptides. Interestingly, the CTLs specific for glycosylated peptides were able to recognize both the Tn (GalNAc) and peptide epitopes. Thus, MUC1 peptides with carbohydrate at the appropriate position may be an optimal vaccine candidate because they can generate CTLs of dual specificity and high affinity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2403.

Abstract It has long been known that immune suppression is induced by the presence of a tumor, resulting in compromised T cell and dendritic cell (DC) functionality. What is not well understood is how soon the immune system recovers normal functionality following surgical removal of tumors and adjuvant therapy. This timing is of critical importance, as the effectiveness of therapeutic immune strategies relies on optimal presentation of antigens and activation of T cells. Blood from breast cancer patients was collected prior to surgery and at 3 and 6 months post adjuvant therapy. Patients were treated with surgery (breast conservation or mastectomy) and chemotherapy (CT) (n=22) or surgery without chemotherapy (n=38). Most patients had radiation therapy (RT). Blood from 22 healthy woman of similar age served as controls. T cell functionality (TCF) was determined following stimulation with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) and proliferation was measured by 3H-thymidine uptake. DC functionality (DCF) was determined by ability to present allo-antigens in a mixed lymphocyte reaction. Values for the normal samples determined the normal ranges (NR). Responses were grouped into 4 categories based on their status at 6 months: Group 1 - remained in NR; Group 2 - climbed into NR; Group 3 - remained below NR; Group 4 - fell below NR. Analysis of immunosuppression will be based on lymphocyte subsets, cytokine, COX-2, and PGE2 metabolite levels.T Cell Proliferation AllWith CTNo CT N=60N=22N=38Group 120%14%24%Group 223%23%24%Group 338%41%37%Group 418%23%16% DC Function AllWith CTNo CT N=58N=20N=38Group 143%35%47%Group 219%25%16%Group 317%15%18%Group 421%25%18% Forty-three percent of patients had normal levels (Groups 1 and 2) for TCF and 62% for DCF by 6 months post adjuvant therapy, suggesting that breast cancer patients are potentially amenable to vaccine therapy. 28% of patients had both TCF and DCF in NR; 50% had either TCF or DCF in NR; and 22% had neither in NR. The percentage of patients who had TCF in NR by adjuvant therapy was: 35% CT+RT (6/17); 33% CT (1/3); 52% (17/33) RT; and 20% (1/5) neither. The percentage of patients who had DCF in NR by adjuvant therapy was: 67% (10/15) CT+RT; 33% (1/3) CT; 67% (22/33) RT; and 40% (2/5) neither. Proper understanding of the effects of tumor, chemotherapy and radiation therapy on immune function, especially the effects on T cells and DCs, may enable us to identify the appropriate patients in whom to study immunotherapy approaches in women with early breast cancer and to examine strategies to counteract cancer-related defects in immune function. (supported by P50CA116201) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4131.