Invention for Peptides, and combinations of peptides, for use in immunotherapy to treat epithelial cancers and other cancers.

Invented by Heiko Schuster, Janet PEPER, Philipp Wagner, Hans-Georg Rammensee, Immatics Biotechnologies GmbH

The Immatics Biotechnologies GmbH invention works as follows

The invention concerns peptides and proteins as well as cells that can be used in immunotherapeutic procedures. The present invention is particularly relevant to immunotherapy for cancer. The invention also relates to tumor-associated T cell peptide epitopes. These can be used alone or in combination, to stimulate immune responses or T cells ex-vivo for transplantation into patients. Antibodies, soluble T cell receptors and other binding molecules can target peptides bound to the major histocompatibility complicated (MHC) or peptides in general.

Background for Peptides, and combinations of peptides, for use in immunotherapy to treat epithelial cancers and other cancers.

Epithelial Ovarian Cancer (EOC) is the leading cause for death from gynecologic cancers in western countries, with an estimated 22,000 new cases diagnosed and 14,000 deaths reported in the US alone in 2014.

The only curative option available is complete surgical removal of the tumor at an early, non-metastatic stage. Most patients (>70%) have stage III or 4 disease due to a lack early symptoms. “Despite advances in chemotherapy regimens, and the recent approval for bevacizumab as first-line therapy, most patients experience relapses within a few months or even years of initial treatment.

There is a pressing need to identify factors which can be used for the treatment of ovarian and cancers in general. It is important to identify biomarkers that can be used to diagnose cancer and to predict treatment outcomes.

Immunotherapy is a way to target cancer cells specifically while minimizing the side effects. Cancer immunotherapy relies on the presence of tumor-associated antigens. “The current classification of TAAs comprises the following major categories:

a) Cancer-testis antigens: The first TAAs ever identified that can be recognized by T cells belong to this class, which was originally called cancer-testis (CT) antigens because of the expression of its members in histologically different human tumors and, among normal tissues, only in spermatocytes/spermatogonia of testis and, occasionally, in placenta. These antigens are immunologically tumor specific because the testis cells do not express class II and I HLA molecules. CT antigens include the MAGE family and NY-ESO-1. The majority of differentiation antigens can be found in normal and malignant melanocytes. These proteins, which are not tumor-specific but are still widely used in cancer immunotherapy, are often involved in the biosynthesis of melanin. Some examples include tyrosinase, Melan-A/MART-1, and PSA, which are used to treat prostate cancer or melanoma. Many of the epitopes that are processed and presented by normal tissue may be below the threshold for T-cell recognition. However, their over-expression by tumor cells can trigger a response against cancer by breaking established tolerance. This class of TAAs includes Her-2/neu or survivin. Telomerase and WT1 are also prominent examples. These molecular modifications are sometimes associated with the neoplastic progression or transformation. In general, tumor-specific antigens can induce strong immune reactions without causing autoimmune reactions to normal tissues. These TAAs, however, are only relevant for the specific tumor that they were found in and are not usually shared by many tumors. TAAs that result from abnormal posttranslational modifications can be derived from proteins that are not tumor specific or overexpressed, but still become tumor associated through posttranslational mechanisms primarily active in tumours. This class includes altered glycosylation patterns that lead to novel epitopes within tumors, such as MUC1, or events like protein degradation which can be tumor-specific or not. These proteins include the human papilloma virus type 16 proteins E6 andE7 that are expressed in cervical cancer.

EOC was recognized over the past two decades as an immunogenic tumor based on a variety of clinical findings. EOC, which showed frequent immune cell invasion, was one of the first cancers where an association between T-cell infiltration (and clinical outcome) could be established. In this population of infiltrating cells, tumor-reactive and antigen-specific T-cells were identified. In contrast, tumor resident regulatory T cells (Tregs), are negatively correlated to clinical outcome. Immune stimulatory cytokines can also induce powerful tumor responses.

The recent approval and development of immune checkpoint inhibitors in the treatment of melanoma has demonstrated that immunotherapeutic cancer therapies are effective. Antigen-specific peptide vaccines and adoptive T cell transfer are also showing success against melanoma, and other immunogenic tumours. Renal cell carcinoma. “Personalized immunotherapy has the potential to cure patients and amazing results have been shown for each patient.

T-cell-based immunotherapy targets epitopes of peptides derived form tumor-associated proteins or tumor-specific protein molecules. These epitopes are presented by molecules in the major histocompatibility complicated (MHC). The tumor-specific T lymphocytes recognize antigens. These epitopes can be molecules from all protein classes such as enzymes and transcription factors. These antigens are usually expressed in tumor cells and up-regulated when compared to cells unaltered of the same origin.

There are two types of MHC-molecules: MHC class I or MHC class 2. MHC class I molecules are composed of an alpha heavy chain and beta-2-microglobulin, MHC class II molecules of an alpha and a beta chain. The binding groove is formed by their three-dimensional conformation, which allows for non-covalent interaction of peptides.

Most nucleated cells contain MHC class I molecules. They contain peptides derived from the proteolytic cleavages of endogenous proteins and defective ribosomal product (DRIP) as well as larger peptides. However, peptides from exogenous or endosomal sources can also be found on MHC class 1 molecules. In literature, this non-classical method of class I display is called cross-presentation (Brossart & Bevan, 1997, Rock et. al., 1990). MHC class II molecules are found primarily on professional antigen-presenting cells (APCs) and present peptides from exogenous or membrane proteins. “During endocytosis and subsequent processing.

Complexes of peptide and MHC class I are recognized by CD8-positive T cells bearing the appropriate T-cell receptor (TCR), whereas complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper-T cells bearing the appropriate TCR. It is well-known that the TCR and the peptide are present in a stoichiometric ratio of 1:1.

CD4-positive T-cell helper cells play an essential role in sustaining and inducing effective responses by CD8+ cytotoxic T-cells. It is important to identify CD4-positive T cell epitopes that are derived from tumor-associated antigens (TAA). This will help in the development of pharmaceuticals for anti-tumor immunity (Gnjatic and colleagues, 2003). At the tumor, T helper cell support a cytotoxic-T-cell-friendly cytokine environment (Mortara, et. al., 2006), and attract effectors, such as. CTLs and natural killer cells (NK) as well as macrophages and granulocytes are all involved in the process of destroying cancerous cells (Hwang et. al., 2007).

In the absence of inflammation the expression of MHC class 2 molecules is restricted to immune cells. This includes monocytes, monocyte derived cells, macrophages and dendritic cell. Cells of tumors have been observed to express MHC Class II molecules in cancer patients (Dengjel, et. al., 2006.)

The invention claims that “Elongated (longer peptides) of the invention may act as MHC Class II active epitopes.

MHC class II epitopes activate T-helper cells and play an important role in the orchestration of CTLs’ anti-tumor immunity effector function. The T-helper cells that activate a T-helper response of the type TH1 support effector functions for CD8-positive killer cells. These functions include cytotoxic functions directed at tumor cells with tumor-associated protein/MHC complexes. This allows tumor-associated Thelper cell peptide epitopes to be used as active pharmaceutical ingredients in vaccine compositions that stimulate antitumor immune responses.

It was demonstrated in mammalian animal models (e.g. mice) that CD4-positive cells can inhibit the manifestation of tumors via inhibition o giogenesis by secretion interferon-gamma. Mumberg et. al. 1999; Beatty and Paterson 2001. Evidence supports the direct anti-tumor effects of CD4 T cells (Braumuller and Paterson, 2013; Tran and al. 2014).

HLA class II constitutive expression is typically limited to immune cells so the possibility of isolating class 2 peptides directly in primary tumors was not previously considered. However, Dengjel et al. Dengjel et al.

Both CD8- and CD4-dependent responses contribute together and synergistically in the anti-tumor effect. It is crucial to identify and characterize tumor-associated antigens by either CD8+ T cell (ligand : MHC Class I molecule+peptide Epitope) or CD4-positive T-helper (ligand : MHC Class II molecule+peptide Epitope) for the development of vaccines against tumors.

For an MHC Class I peptide (to trigger or elicit) an immune response in the cell, it must also bind to an MHC molecule. This depends on the MHC-molecule allele and the specific polymorphisms in the amino acid sequence. MHC-class I-binding amino acids are typically 8-12 residues long and contain two conserved (?anchors?) In their sequence, they interact with the binding grooves of the MHC-molecule. Each MHC allele thus has a “binding motif” “determining which peptides are able to bind specifically to binding groove.

In order to activate the MHC Class I dependent immune reaction, peptides must not only be able to bind certain MHC class I molecules produced by tumor cells but also need to be recognized by T cells that have specific T cell receptors (TCR).

For proteins to be recognized as tumor-specific antigens or antigens associated with tumors by T-lymphocytes and used in therapy, certain prerequisites must be met. The antigen must be expressed by the tumor cells in large amounts and by healthy normal tissues only in small quantities. In a preferred embodiment the peptide is over-expressed by tumor cells compared with normal healthy tissue. Further, it is desirable that not only is the antigen present in certain types of tumors but also at high concentrations. The peptide should be present in high concentrations (i.e. Antigens that are tumor-specific or tumor-associated are frequently derived from proteins involved in the transformation of normal cells into tumor cells due to their functions, e.g. Cell cycle control and suppression of apoptosis are two examples. Furthermore, downstream targets for the proteins that are directly responsible for a mutation may also be up-regulated and thus indirectly tumor-associated. These indirect antigens can also be used as targets for a vaccine (Singh Jasuja and colleagues, 2004). In order for a peptide to be considered an immunogenic peptide, it is important that epitopes appear in the amino-acid sequence of the antigen. The peptide, which is derived from an antigen associated with tumors, can lead to a T-cell response in vitro or in-vivo.

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