Invented by Eric POMA, Erin WILLERT, Molecular Templates Inc
The Molecular Templates Inc invention works as followsThe present invention relates to T-cell Epitope-Delivering Polypeptides that deliver one or more CD8+T-cell epitopes to a cell’s MHC class I presentation pathway. This includes toxin-derived polypeptides that contain embedded T-cell epitopes, and are de-immunized. Cell-targeted CD8+ T cell epitope delivery molecules are provided by the present invention for the targeted delivery cytotoxicity to specific cells, e.g. infected or malignant cell lines, and treatment of various diseases, disorders and conditions including cancers, immune disorders and microbial infections. The present invention provides methods for generating polypeptides capable to deliver one or more heterologous epitopes to T-cells through the MHC class 1 presentation pathway. This includes polypeptides that are 1) B-cell or CD4+ T cell de-immunized, 2) contain embedded T-cell epitopes and/or (3) comprise toxin effectors that retain toxin function.
Background for MHC class I epitope delivering polypeptides
The immune systems of chordates (amphibians, birds, fishes, mammals, reptiles and sharks) constantly monitor the extracellular as well as intracellular environments for exogenous chemicals in an effort to detect the presence of potentially dangerous foreign molecules, cells, or pathogens. As part of adaptive immunity, chordates have the Major Histo-Compatibility system (MHC). Janeway’s Immunobiology (MurphyK, ed. Garland Science 8th edition, 2011). Extracellular antigens in a chordate are presented by MHC class II, while intracellular antigens may be presented by MHC Class I.
Exogenous peptides or polypeptides are not allowed to enter cells due to the physical barrier created by the plasma membrane. These molecules can also be broken down into smaller molecules through extracellular enzymes on cells’ surfaces and/or in extracellular milieu. Endocytosis is a process that removes proteins and polypeptides from the extracellular environment. Lysosomal proteolysis is a part of an endocytotic pathway that involves early, late, and lysosomes. The same pathway that ends with phagolysosomes is used to degrade proteins and polypeptides that are phagocytosed from the extracellular environment.
The MHC Class II pathway contains antigenic peptides that are derived from molecules in extracellular space. These peptides are processed by antigen-presenting cells. They can be professional antigen-presenting cells or other antigen-presenting cells such as dendritic cell (DCs), mononuclear (MNPCs), certain endothelial and B-lymphocytes. Antigen-presenting cells have certain peptides that are complexed with MHC II molecules on the cell surface to be recognized by CD4+ (CD4+), T-lymphocytes. The MHC class 1 system, on the other hand, is used in chordate cells to present antigenic propeptides from intracellular space (most commonly the cytosol) for CD8+ T cell recognition.
The MHC Class I system plays an important role in the immune response by providing intracellular antigens. (Cellular and Molecular Immunology, Abbas A, ed. Saunders, 8th ed. 2014). This is believed to be an important part the adaptive immune system that evolved in chordates to protect against neoplastic and microbial infections involving intracellular Pathogens. However, it can also be used to remove damaged cells. An antigenic peptide is a peptide that has been combined with an MHC class I molecule. This makes it possible for the cells to be targeted killed by cytotoxic T cell (CTLs). The process can be performed via lysis, inducing apoptosis and/or necrosis. Specific peptide epitopes that are complexed with MHC Class I molecules play a significant role in stimulating and maintaining immune response to tumors, cancers, and intracellular pathogens.
The MHC Class I system continuously processes and displays on the cell surface various intracellular Epitopes, either self or foreign (foreign), and both peptide and lipid antigens. The MHC class 1 display of foreign antigens, whether from intracellular pathogens and transformed cells, signals to CD8+ effector-T-cells to mount protective immune responses. The MHC class 1 system also functions continuously to present self-peptide epitopes to maintain and establish immunological tolerance.
Peptide epitope presentation via the MHC Class I system involves five major steps: 1) generation and transport of cytoplasmic peptides to ER, 2) stable complex formation MHC class 1 molecules bound to specific peptides, 3) display of those stable peptide -MHC molecule complexes (peptide?MHC class I complexes on the cell surface), and 5) recognition by certain CD8+ T cells, including CTLs, certain antigenic, presented peptides
A CD8+T-cell recognizes a present antigen-MHC Class I complex and activates CD8+ T cells. This leads to clonal expansion and differentiation into CD8+ effector cell types, including CTLs that target cells with specific epitopes-MHC Class I complexes. This creates a population CD8+ effector cell, which can travel throughout your body to search for and destroy cells that have a specific epitope.
A cytosolic protein is used to initiate the MHC class I system. There are many ways that peptides can exist in the cytosol. MHC class I molecules generally present peptides that are derived from proteasomal degrading intracellular proteins and polypeptides. Transporters that are associated with antigen processing protein (TAPs), which are associated with the ER membrane, can be used to initiate the MHC class I pathway. TAPs move peptides from cytosol to the lumen ER where they can associate with empty MHC Class I molecules. TAPs can translocate peptides that are most common sizes between 8-12 amino acids residues, but also include 6-40 amino acids residues (Koopmann J. et al. Eur J Immunol26: 1720-8 (1996 )).”).
The MHC-class I pathway can also start in the lumen ER. It involves transporting a protein, polypeptide or peptide into cytosol for processing, and then re-entry into the ER via TAPmediated translocation.
A CD8+ T cell recognizes an epitope of MHC class I complex. This initiates protective immune reactions that ultimately end in the death or cytotoxic activity of one of several CTLs. CTLs have different T-cell receptors, (TCRs), with different specificities. These MHC alleles vary greatly and can have an impact on T-cell recognition in two ways. They can alter the binding of peptides or the region between TCRs and MHC molecules. The CTL will respond to antigen-MHC molecule complex recognition by its specific cell surface TCR and kill the antigen -MHC molecule complex presenting cells primarily through cytolytic activities. This is mediated by the injection of perforin or granzyme into presenting cells. The CTL can also release immuno-stimulatory cytokines such as interferon gamma, tumor necrosis factor beta (TNF), macrophage inflammatory proteins-1 beta (MIP-1beta), as well as interleukins like IL-17, IL-4 and IL-22. CTLs activated can kill any proximal epitope-MHC complex presenting cells, regardless of their peptide-MHC complex repertoire. Therapeutics could harness these epitope-MHC complex-induced immune responses to kill specific cell types within patients and also to sensitize the immune systems to other proximal ones.
The MHC-class I presentation pathway could potentially be exploited for therapeutics to induce desired immune reactions. However, there are many barriers to such a technology being developed, including delivery through the cell plasma membrane, escape from the endocytotic pathway, destruction in the Lysosome, and avoiding sequestration modification and/or destruction foreign polypeptides by the targeted cells (Sahay G. et.al., J Control Release.145: 182-195 (2010); Fuchs h., Antibodies 2.
In addition, the effectiveness of polypeptide-comprising therapeutics, e.g. “Polypeptide-based biologics and biopharmaceuticals are often limited by the undesirable immune reactions generated in patients in response to therapeutics. Nearly all polypeptide-based therapies induce an immune response when administered to mammalian subjects. There are many levels of immune response, from low-level, low affinity and transient immunoglobulin M antibodies to high-level high-affinity immunoglobulin G antibodies. Unwanted immune responses to a therapeutic could reduce therapeutic efficacy, adversely affect pharmacokinetics and/or cause hypersensitivity reactions, anaphylaxis or infusion reactions. (See Buttel I et. al., Biologicals39:100-9 (2011 )).”).
A polypeptide-based therapeutic, for example, can cause a recipient’s immune system to produce antibodies against antigenic site(s) in the therapeutic (sometimes known as anti-drug or neutralizing antibodies). Immune reactions that generate antibodies to recognize a therapeutic may result in immunological resistance. Cross-reactions between antitherapeutic antibodies and endogenous factors may also lead to undesirable clinical outcomes.
Polypeptide-based therapies with polypeptides sequences derived form species distantly related (e.g. when the recipient is a mammal) tend to be aggressively targeted and regulated by the recipient’s immune systems (see Sauerborn M. et.l., Trends Pharmacol Sci 31, 53-9 (2010)). The immune systems of vertebrates have evolved to recognize foreign polypeptides using both their adaptive and innate immune systems. Thud, administration of a protein to a vertebrate of the same species can be recognized as nonself and elicit an immune reaction, such as administering to a human a mixture of two heterologous human-derived polypeptide sequences.
Therefore, when designing polypeptide-containing therapeutics it is often desirable to attempt to minimize the immunogenicity of the therapeutic to prevent and/or reduce the occurrence of undesired immune responses in subjects undergoing therapeutic treatment. It is important to target polypeptide areas in therapeutics that are likely to cause B-cell, T-cell, or immunogenicity, as these regions should be eliminated, suppressed, and minimized.
Both T-cell and B-cell epitopes can both be predicted in a given sequence of polypeptides in silico using software” (Bryson C. et al. BioDrugs 24: 1-8 (2010). Software called EpiMatrix (EpiVax, Inc., Providence, R.I., U.S.A.) was used to successfully predict T-cell immunogenicity using recombinant proteins. (De Groot A. et., Dev Biol. 122: 171?94 (2005); Koren E. et., Clin Immunol. 124: 26?32 (2007 )).”).
Many approaches, such as the elimination of antigenic and/or immunogenic epitopes by truncation or mutation, have been described for reducing the immunogenicity of polypeptide-containing therapeutics (Tangri S et al., J Immunol 174: 3187-96 (2005); Mazor R et al., Proc Natl Acad Sci USA 109: E3597-603 (2012); Yumura K et al., Protein Sci 22: 213-21 (2012)). The adaptive immune system can recognize foreign polypeptides with remarkable specificity. This is because immune epitopes are often found at very few sites on the polypeptide’s surface. Interactions with a few specific amino acids in an epitope can alter antibody-binding affinity. Modifications of key amino acids within a polypeptide that disrupt an immunogenic epitope may reduce immunogenicity (LarocheY et.al., Blood96: 1425-32, 2000). Amino acid substitutions, amino acid deletions, and epitope-mapping with non-immunogenic conjugates are all possible ways to disrupt epitope recognition.
It is important to avoid inducing immune responses in B-cells and the production neutralizing antibodies in patients in order to develop polypeptide-based therapeutics (see Lui W. et al. Proc Natl Aca Sci USA 109, 11782-7 (2012 )).”).
It would be beneficial to have novel methods for creating T-cell epitope-delivering polypeptides that can deliver T-cell epitopes to a cell’s MHC class I presentation pathway. Polypeptides that can deliver a T cell epitope to the target cell’s interior under physiological conditions would be desirable. However, they do not induce unwanted immune responses in extracellular spaces such as the formation of inhibitory antibodies. It would be desirable to have polypeptides that deliver T-cell epitopes to T-cells, in which one or two CD8+ T cell epitopes and one or both of the CD4+ T.cell epitopes is removed.
It would be nice to have cell-targeted CD8+ T cell epitope delivering molecular for targeted delivery of cytotoxicity, e.g. infected cells or malignant cells. It would also be beneficial to have cell-targeted CD8+ T?cell epitope delivery molecules that exhibit lower B-cell immunogenicity. The T-cell immunogenic protein(s) delivered to the target cell’s surface by the cell-targeted molecular can activate the recipient’s immune system to recruit more CD8+ T cells. The target cell’s T-cell epitope MHC class I complex, which activates CD8+ T cells, can trigger a larger immune response and alter micro-environment (e.g. Release cytokines from a tumor or infected tissue locus, so that other immune cells (e.g. “Effector T-cells” may be recruited to the area.Click here to view the patent on Google Patents.