BACKGROUND OF THE INVENTION

Field of the Invention

This application relates generally to modified, non-natural α1-α2 domains of NKG2D ligands with attached polypeptides having specific target-binding properties, for example, antibodies or fragments of antibodies, that are selectively delivered to Chimeric Antigen Receptors (CARs) comprised of modified, non-natural NKG2D receptors on engineered mammalian cells.

Background Information

An antibody (Ab),FIG. 1, also known as an immunoglobulin (Ig), in many mammals including humans is a large, Y-shape protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses (Charles Janeway (2001).Immunobiology.(5th ed.), Chapter 3. Garland Publishing. ISBN 0-8153-3642-X. (electronic full text via NCBI Bookshelf). The antibody recognizes a unique part of the foreign target, called an antigen. Each tip of the two arms of the “Y” of an antibody contains an antigen binding site, or a paratope, (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) of an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system or can neutralize its target directly, for example, by blocking a part of a microbe that is essential for its invasion and survival. The production of antibodies is the main function of the humoral, or “adaptive”, immune system. Antibodies are secreted by plasma cells. Antibodies in nature can occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell via the “stem” of the Y.

Antibodies are glycoproteins belonging to the immunoglobulin superfamily and are typically made of basic structural units—each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals (Market E, Papavasiliou F N (October 2003). “V(D)J recombination and the evolution of the adaptive immune system”.PLoS Biol.1 (1): E16. doi:10.1371/journal.pbio.0000016. PMC 212695. PMID 14551913). Although the general structure of all antibodies is very similar, a small region at the tip of each arm of the Y-shaped protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen-binding sites, to exist. This region is known as the hypervariable or variable region. Each of these natural variants can bind to a different antigen. This enormous diversity of antibodies allows the immune system to adapt and recognize an equally wide variety of antigens (Hozumi N, Tonegawa S (1976). “Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions”.Proc. Natl. Acad. Sci. U.S.A.73 (10): 3628-3632. doi:10.1073/pnas.73.10.3628. PMC 431171. PMID 824647.)

The natural “Y”-shaped Ig molecule consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. Each heavy chain has two major regions, the constant region (CH) and the variable region (VH). The constant region is essentially identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. A light chain also has two successive domains: a smaller constant region (CL) and the variable region (VL) (Woof J, Burton D (2004). “Human antibody-Fc receptor interactions illuminated by crystal structures.”Nat Rev Immunol4 (2): 89-99. doi:10.1038/nri1266. PMID 15040582).

Some parts of an antibody have the same functions. Each of the two arms of the Y, for example, contains the sites that can bind to antigens and, therefore, recognize specific foreign objects. This region of the antibody is called the Fv (fragment, variable) region. It is composed of one variable domain from the heavy chain (VH) and one variable region from the light chain (VL) of the antibody (Hochman J, Inbar D, Givol D (1973). An active antibody fragment (Fv) composed of the variable portions of heavy and light chains. Biochemistry 12 (6): 1130-1135. doi:10.1021/bi00730a018. PMID 4569769). The paratope is shaped at one end of the Fv and is the region for binding to antigens. It is comprised of variable loops of β-strands, three each on the VLand on the VHand is responsible for binding to the antigen. These 6 loops are referred to as the complementarity determining regions (CDRs) (North B, Lehmann A, Dunbrack R L (2010). “A new clustering of antibody CDR loop conformations”.J Mol Biol406 (2): 228-256. doi:10.1016/j.jmb.2010.10.030. PMC 3065967. PMID 21035459).

Useful polypeptides that possess specific antigen binding function can be derived from the CDRs of the variable regions of antibodies. These two antibody variable domains, one of the light chain (VL) and one from the heavy chain (VH), each with 3 CDRs can be fused in tandem, in either order, using a single, short linker peptide of 10 to about 25 amino acids to create a linear single-chain variable fragment (scFv) polypeptide comprising one each of heavy and light chain variable domains (Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S., and Whitlow, M. (1988) Single-chain antigen-binding proteins, Science 242, 423-426; Huston, J. S., Levinson, D, Mudgett-Hunter, M, Tai, M-S, Novotny, J, Margolies, M. N., Ridge, R., Bruccoleri, R E., Haber, E., Crea, R., and Opperman, H. (1988). Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced inEscherichia coli. PNAS 85: 5879-5883).

The linker is usually rich in glycine for flexibility, as well as serine, threonine, or charged amino acids for solubility, and can either connect the N-terminus of the VHwith the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the single linker. This format enables one ordinarily skilled in the art of recombinant DNA technology to genetically fuse the linear scFv to the N- or C-terminus of a parent protein in order to impart to the parent protein the antigen binding properties of the scFv. There are numerous other proposed or created arrangements of polyvalent and tandem scFv regions, but importantly as described below, all have at least two spatially distant termini, (Le Gall, F.; Kipriyanov, S M; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding”. FEBS Letters 453 (1): 164-168. doi:10.1016/S0014-5793(99)00713-9. PMID 10403395).

SUMMARY OF THE INVENTION

The present disclosure relates to modified α1-α2 domains of NKG2D ligands attached to heterologous polypeptides, in some embodiments antibodies or fragments of antibodies. The modified ligands bind selectively to cognate non-natural NKG2D receptors which in-turn bind selectively to their cognate modified ligands. The non-natural NKG2D receptors can be expressed on the surfaces of cells of the immunity system and create a chimeric receptor on surface of that effector cell. The heterologous molecule attached to the ligand may also bind a specific molecule on the surface of a target cell, thereby delivering the immunity effector cell to a target cell. Such effector cells include lymphocytes, B-cells, plasma cells, monocytes, macrophages and dendritic cells.

In some embodiments, the present disclosure relates to a modified, non-natural ligand for a modified, non-natural NKG2D receptor wherein the ligand has an attached heterologous molecule that selectively binds an HIV protein present on the surface of a cell infected by HIV, wherein the modified ligand with its heterologous molecule can selectively bind to a modified, non-natural NKG2D receptor of a CAR-cell and cause the destruction of the HIV-infected cell.

In further embodiments, the HIV protein which the heterologous molecule selectively binds to is an HIV envelope protein.

In yet further embodiments, the epitope of the envelope protein to which the heterologous molecule selectively binds comprises SEQ ID NO: 169 or SEQ ID NO: 170.

In some embodiments of the present disclosure, the modified, non-natural ligand comprises SEQ ID NO: 68, 69, 70, 71, or 72.

In further embodiments, the modified, non-natural NKG2D receptor comprises SEQ ID NO: 54 or 154.

In some embodiments of the present disclosure, the HIV protein is expressed on an HIV infected cell that has been shocked or activated by a mechanism or agent, or a latency reactivating agent, known to provoke the expression of an HIV protein on a latent HIV-infected cell.

In some embodiments of the present disclosure, the CAR-cell has bound multiple modified, non-natural ligands with different, distinct heterologous molecules binding different epitopes, proteins or other molecules on the surface of an HIV-infected cell.

In some embodiments of the present disclosure, a modified, non-natural NKG2D receptor comprising SEQ ID NO: 54 or 154 is present on a CAR-cell, wherein the modified NKG2D receptor binds a modified, non-natural ligand comprised of SEQ ID NO: 68, 69, 70, 71, or 72, to which is attached a heterologous molecule or atom that does not bind an HIV protein.

In further embodiments, the heterologous molecule or atom modulates the function of the CAR-cell. In yet further embodiments, the cellular function includes proliferation, differentiation, ablation, imaging, antagonism of immunosuppression, homing, or cytolysis of a cell not infected by HIV.

