CD8 binding agents

#The present invention relates, in part, to binding agents (e.g., antibodies, such as, without limitation, VHHs) which bind CD8 and their use as therapeutic and diagnostic agents.

#The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: ORN-011PC_Sequence_listing date recorded: Feb. 1, 2017; file size: 265 KB).

#Despite major advances in medicine, cancer remains one of the leading causes of death globally, with an estimated 12.7 million cases each year. One of the major stumbling blocks in designing effective anticancer therapy is cancer immune evasion, in which cancer cells evade immune surveillance and destruction thereby resulting in clinically overt cancer. Mechanisms of immune evasion include the selection of tumor variants resistant to immune effectors and the progressive formation of an immune suppressive environment within the tumor.

#FIG. 1, panel A, provides histograms showing the binding of six VHHs specific for mouse CD8 to CHO cells transfected with CD8a. Panel B provides histograms showing mouse splenocytes stained with six VHHs specific for mouse CD8. The relative binding affinities of the six VHHs are as follows: I-11269>I-11265 and I-11268>I-11278 and I-11287.

#FIG. 2 provides histograms depicting the effects of various VHHs specific for mouse CD8 on OVA-induced T cell proliferation and activation.

#FIG. 3 shows that the administration of a fusion of a VHH specific for mouse CD8 with a modified human interferon (Q124R mutant) led to reductions in tumor size. mCD8Nb(R1) corresponds to clone R1CDE28 and mCD8Nb(R2) corresponds to clone R2CDE71.

#The present invention is based, in part, on the discovery of agents (e.g. antibodies such as, by way of non-limiting example, VHHs) that recognize and bind to CD8. In various embodiments, these CD8 binding agents bind to, but do not functionally modulate CD8. In various embodiments, these CD8 binding agents may bind and directly or indirectly recruit immune cells to sites in need of therapeutic action (e.g. a tumor). The present invention provides pharmaceutical compositions comprising the CD8 binding agents and their use in the treatment of various diseases.

#CD8 Binding Agents

#In various embodiments, the present CD8 binding agent is a protein-based agent capable of specific binding to CD8. In various embodiments, the present CD8 binding agent is a protein-based agent capable of specific binding to CD8 without functionally modulation (e.g. partial or complete neutralization) of CD8. CD8 is a heterodimeric type I transmembrane glycoprotein, whose α and β chains are both composed of an immunoglobulin (Ig)-like extracellular domain connected by an extended O-glycosylated stalk to a single-pass transmembrane domain and a short cytoplasmic tail (Li et al., 2013). The cytoplasmic region of the α-chain contains two cysteine motifs that serve as a docking site for src tyrosine kinase p56lck (Lck). In contrast, this Lck binding domain appears to be absent from the β chain, suggesting that the CD8 β chain is not involved in downstream signaling (Artyomov et al., 2010). CD8 functions as a co-receptor for the T-cell receptor with its principle role being the recruitment of Lck to the TCR-pMHC complex following co-receptor binding to MHC (Turner et al., 1990, Veillette et al., 1988). The increase in the local concentration of this kinase activates a signaling cascade that recruits and activates ζ-chain-associated protein kinase 70 (ZAP-70), subsequently leading to the amplification or enhancement of T-cell activation signals (Purbhoo et al., 2001, Laugel et al., 2007a).