T-cell modulatory multimeric polypeptide with conjugation sites and methods of use thereof
The present disclosure provides T-cell modulatory multimeric polypeptides (“T-Cell-MMPs”) comprising an immunomodulatory polypeptide (“MOD”) that may be selected to exhibit reduced binding affinity to a cognate co-immunomodulatory polypeptide (“Co-MOD”) and a location for covalently attaching a molecule that can serve as an epitope, such as an epitope peptide. Once the epitope molecule is attached the resulting T-Cell-MMP-epitope conjugates are useful for modulating the activity of a T-cell by delivering immunomodulatory peptides, such as IL-2 or IL-2 variants that exhibit reduced binding affinity for IL-2R, to the T-cells in an epitope selective/specific manner, and accordingly, for modulating an immune response in an individual.
1. A T-cell modulatory multimeric polypeptide (T-Cell-MMP) comprising:
a) a first polypeptide comprising,
i) a first major histocompatibility complex (MHC) polypeptide having an N-terminus and a C-terminus, wherein the first MHC polypeptide comprises a beta-2-microglobulin (β2M) polypeptide;
b) a second polypeptide having an N-terminus and a C-terminus comprising, in order from N-terminus to C-terminus,
(i) a second MHC polypeptide, wherein the second MHC polypeptide comprises a class I MHC heavy chain polypeptide comprising a binding pocket;
(ii) optionally a peptide linker; and
(iii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig polypeptide scaffold;
c) (i) a first polypeptide chemical conjugation site that is part of a linker attached to the first polypeptide or is within the first polypeptide, or
(ii) a second polypeptide chemical conjugation site that is part of a linker attached to the second polypeptide or is within the second polypeptide;
wherein the first and second polypeptide chemical conjugation sites are sites at which a molecule comprising an epitope peptide may be covalently bound, either directly or indirectly through a linker, to the first or second polypeptide and positioned in the binding pocket of the T-Cell MMP for presentation to a cell bearing a T-cell receptor specific for the epitope presented by the epitope peptide; and
d) one or more immunomodulatory polypeptides (MODs), wherein at least one of the one or more MODs is
i) at the C-terminus of the first polypeptide,
ii) at the N-terminus of the second polypeptide,
iii) at the C-terminus of the second polypeptide, or
iv) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide;
wherein each of the one or more MODs is an independently selected wild-type or variant MOD.
2. The T-Cell-MMP of claim 1 , wherein the second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide or a non-Ig polypeptide scaffold.
3. The T-Cell-MMP of claim 1 , wherein the T-Cell-MMP comprises one or more independently selected variant MOD polypeptides; wherein at least one of the one or more variant MOD polypeptides exhibits a reduced affinity to its Co-MOD compared to the affinity of a corresponding wild-type MOD for the Co-MOD; and wherein the ratio of i) the binding affinity of a control T-Cell-MMP-epitope conjugate to a Co-MOD to ii) the binding affinity of a T-Cell-MMP-epitope conjugate comprising a variant of the wild-type MOD to the Co-MOD, when measured by bio-layer interferometry (“BLI”), is at least 1.5:1 or in a range of from 1.5:1 to 10 6 :1.
4. The T-Cell-MMP of claim 1 , wherein the wild-type MOD polypeptides are selected independently from the group consisting of IL-2, 4-1BBL, PD-L1, CD70, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1, TGFβ, ICAM, and PD-L2, and the variant MOD polypeptides are variants thereof.
5. The T-Cell-MMP of claim 1 , wherein the first or second chemical conjugation site is independently selected from:
peptide sequences that act as an enzymatic modification sequence;
non-natural amino acids and/or selenocysteines;
engineered amino acid chemical conjugation sites;
carbohydrate or oligosaccharide moieties; and/or
IgG nucleotide binding sites.
6. The T-Cell-MMP of claim 5 , wherein the first or second chemical conjugation site comprises an engineered amino acid chemical conjugation site.
7. The T-Cell-MMP of claim 6 , wherein the engineered amino acid chemical conjugation site comprises a cysteine.
8. The T-Cell-MMP of claim 7 , wherein: the cysteine of the chemical conjugation site is present in the β2M polypeptide sequence, and said β2M polypeptide sequence has at least 90% amino acid sequence identity to amino acids 21-119 of any one of SEQ ID NOs: 151-155; and said class I MHC heavy chain polypeptide has at least 90% sequence identity to at least 250 contiguous amino acids of an MHC heavy chain sequence set forth in any one of SEQ ID NOs: 134-150.