EA042111B1 - METHODS FOR MODULATING THE IMMUNE RESPONSE - Google Patents

Description

cross reference

This application claims priority over U.S. Provisional Application No. 62/471,832, filed March 15, 2017, and U.S. Provisional Application No. 62/521,009, filed June 16, 2017, which applications are incorporated herein in their entirety by links.

Introduction.

The adaptive immune response involves the interaction of a T cell receptor (TCR) present on the surface of T cells with a small peptide antigen presented non-covalently on the surface of an antigen presenting cell (APC) by a major histocompatibility complex. (MCGS; also referred to in humans as the human leukocyte antigen (HLA) complex). This interaction is the targeting mechanism of the immune system and is a necessary molecular interaction for T cell modulation (activation or inhibition) and effector function. After epitope-specific targeting of target cells, T cells are activated through the interaction of co-stimulatory proteins found on APC with analogs of co-stimulatory proteins of T cells. Both signals - epitope binding to TCR and interaction of APC costimulatory proteins with T cell costimulatory proteins - are required to control specificity and activation or inhibition of T cells. TCR is specific to this epitope; however, the co-stimulatory protein is not specific to an epitope and is instead usually expressed on all T cells or large subgroups of T cells.

The essence of the invention

The invention provides methods for modulating an immune response in an individual. The present disclosure provides methods of treatment. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The present invention relates to methods comprising administering a multimeric polypeptide (synTac) to an individual who is being treated with an immune checkpoint inhibitor.

Brief description of graphic materials

In FIG. 1A-1D schematically depict various embodiments of a modulating T cell multimeric polypeptide. In these embodiments, disulfide bonds are formed between MHC (eg, HLA) polypeptides present in individual polypeptides.

In FIG. 2A-2Q are the amino acid sequences of wild-type human IL-2 (FIG. 2A); and amino acid sequences of variant IL-2 polypeptides (FIGS. 2B-2Q).

In FIG. 3A-3C are the amino acid sequences of the IL-2 receptor alpha chain (FIG. 3A), beta chain (FIG. 3B) and gamma chain (FIG. 3C).

In FIG. 4A-4C are the amino acid sequences of immunoglobulin Fc polypeptides.

In FIG. 5A-5C are the amino acid sequences of human leukocyte antigen (HLA) class I heavy chain polypeptides. Signal sequences are underlined.

In FIG. 6 shows an alignment of multiple amino acid sequences of beta-2-microglobulin precursors (b2M) (i.e. including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 95), Pan troglodytes (NP_001009066.1; SEQ ID NO: 195), Macaca mulatto (NP_001040602.1; SEQ ID NO: 96), Bos taurus (NP_776318.1; SEQ ID NO: 97) and Mus musculus (NP_033865.2; SEQ ID NO: 98). Amino acids 1-20 are the signal peptide.

In FIG. 7A-7B show the production of IL-2/synTac (Cue-IL-2-a and Cue-IL-2-b) of the present invention after transient transfection. In FIG. 7A shows the raw output, FIG. 7B shows the purified product.

In FIG. 8A-8B show the production of IL-2/synTac of the present invention, in which the IL-2 polypeptide is present in the light chain (the light chain polypeptide chain (e.g., B2M) of the MHC class I molecule) or in the heavy chain (heavy chain polypeptide chain MHC class I molecules).

In FIG. 9 shows the expression level of IL-2/syn-Tac in which IL-2 is wild-type (wt) or contains various combinations of F42A, D20K, Q126A, E15A, Y45A and H16A.

In FIG. 10 shows the expression of IL-2/synTac according to the present disclosure, in which IL-2 is present in one copy (1X), two copies (2X) or three copies (3X) in synTac.

In FIG. 11 shows in vitro stimulation of antigen-specific CD8+ T cells and non-specific CD8+ T cells with IL-2/synTac of the present invention, where an IL-2 variant containing F42A and H16A substitutions is present in synTac in two copies.

In FIG. 12 shows binding of IL-2/synTac to specific (lymphocytic choriomeningitis virus; LCMV) or non-specific (OT1; ovalbumin recognizing) CD8+ T cells.

In FIG. 13 shows IL-2/synTac-mediated signaling in antigen-specific (LCMV) or non-specific (BL6) CD8+ T cells.

In FIG. 14A-14F show the percentage of phosphosignal transducer and transcriptional activator 5 (pSTAT5)-positive cells following stimulation of CD8+ antigen-specific (LCMV) or non-specific (BL6) cells with IL-2/synTac of the present invention at various concentrations of IL-2. /synTac.

In FIG. 15 shows the in vivo activity of IL-2/synTac of the present invention. The left panel shows the fold change in the number of antigen-specific CD8+ T cells after administration of phosphate-buffered saline (PBS), recombinant IL-2 (rIL-2) or IL-2/synTac of the present invention. The right panel depicts antigen-specific and non-antigen-specific responses after administration of PBS, rIL-2 or IL-2/synTac of the present invention.

In FIG. 16A-16B show the effects of increasing dose (FIG. 16A) and route of administration (FIG. 16B).

In FIG. 17A-17B show the effect of IL-2 copy number on in vivo efficacy against tumor.

In FIG. 18 shows the serum half-life of IL-2/synTac of the present invention following intraperitoneal administration of IL-2/synTac at 10 mg/kg.

In FIG. 19 shows the stability of IL-2/synTac of the present disclosure 2 hours after intraperitoneal administration of IL-2/synTac at 10 mg/kg.

In FIG. 20 shows the size exclusion chromatography data of IL-2/synTac of the present invention after keeping IL-2/synTac at 4°C or 37°C for 5 days.

In FIG. 21 shows the amino acid sequence of an IL-2/synTac heavy chain of the present invention with a leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with the N297A substitution.

In FIG. 22 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention without the leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with the N297A substitution.

In FIG. 23A-23B show the nucleotide sequence (FIG. 23A) encoding the IL-2/synTac heavy chain shown in FIG. 21; and a key (FIG. 23B) to the sequence.

In FIG. 24 shows the amino acid sequence of an IL-2/synTac heavy chain with a leader peptide, where the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234A and L235A.

In FIG. 25 shows the amino acid sequence of an IL-2/synTac heavy chain without a leader peptide, where the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234A and L235A.

In FIG. 26A-26B show the nucleotide sequence (FIG. 26A) encoding the IL-2/synTac heavy chain shown in FIG. 24; and a key (FIG. 26B) to the sequence.

In FIG. 27 shows the amino acid sequence of an IL-2/synTac heavy chain with a leader peptide, where the IL-2/synTac heavy chain contains IgG1 Fc with substitutions L234F, L235E and P331S.

In FIG. 28 shows the amino acid sequence of an IL-2/synTac heavy chain without a leader peptide, where the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234F, L235E, and P331S.

In FIG. 29A-29B show the nucleotide sequence (FIG. 29A) encoding the IL-2/synTac heavy chain shown in FIG. 27; and a key (FIG. 29B) to the sequence.

In FIG. 30 shows the amino acid sequence of an IL-2/synTac light chain with a leader peptide, wherein the IL-2/synTac light chain contains the human papillomavirus (HPV) E7 epitope.

In FIG. 31 shows the amino acid sequence of the IL-2/synTac light chain without the leader peptide, where the IL-2/synTac light chain contains the HPV E7 epitope.

In FIG. 32 shows the nucleotide sequence encoding the IL-2/synTac light chain shown in FIG. thirty.

In FIG. 33A-33D are the amino acid sequences of wild type human IgG1 Fc (FIG. 33A), IgG1 Fc with L234F, L235E, and P331S substitutions (FIG. 33B), IgG1 Fc with N297A substitution (FIG. 33C), and IgG1 Fc with L234A and L235A substitutions. (Fig. 33D).

In FIG. 34A-34C are the amino acid sequences of the β2-microglobulin (R12C) polypeptide (FIG. 34A), the variant IL-2 polypeptide (H16A; F42A) (FIG. 34B), and the A0201 class I MHC-H chain (Y84A; A236C) (FIG. .34C).

In FIG. 35 shows the synergistic effect of IL-2/synTac and anti-PD1 antibody on tumor volume reduction.

In FIG. 36A-36PP show the amino acid sequence of 4-1BBL (FIG. 36A) and exemplary 4-1BBL polypeptide variants (FIGS. 36B-36IIII).

In FIG. 37 shows the amino acid sequence of 4-1BB.

In FIG. 38A-38B show interferon-gamma (IFN-γ) secretion by target cells contacting a synTac polypeptide for 3 days (FIG. 38A) or 5 days (FIG. 8B) according to an embodiment of the present disclosure.

In FIG. 39A-39B depict interleukin-2 (IL-2) secretion by target cells exposed to a synTac polypeptide for 3 days (FIG. 39A) or 5 days (FIG. 9B), according to an embodiment of the present disclosure.

In FIG. 40A-40B show secretion of interleukin-6 (IL-6) by target cells contacting the synTac polypeptide for 3 days (FIG. 40A) or 5 days (FIG. 40B) according to variant

- 2 042111 volume of implementation of the present disclosure.

In FIG. 41A-41B show secretion of tumor necrosis factor-alpha (TNFa) from target cells contacted with a synTac polypeptide for 3 days (FIG. 41A) or 5 days (FIG. 41B) according to an embodiment of the present disclosure.

In FIG. 42A-42B show secretion of interleukin-10 (IL-10) by target cells contacting a synTac polypeptide for 3 days (FIG. 42A) or 5 days (FIG. 42B) according to an embodiment of the present disclosure.

In FIG. 43A-43B show secretion of interleukin-17A (IL-17A) by target cells exposed to a synTac polypeptide for 3 days (FIG. 43A) or 5 days (FIG. 43B) according to an embodiment of the present disclosure.

In FIG. 44A-44B show secretion of interleukin-4 (IL-4) by target cells contacting the synTac polypeptide for 3 days (FIG. 44A) or 5 days (FIG. 44B) according to an embodiment of the present disclosure.

In FIG. 45 shows the proliferation of target cells contacting the synTac polypeptide according to an embodiment of the present invention.

In FIG. 46 shows the viability of target cells contacting the synTac polypeptide according to an embodiment of the present invention.

In FIG. 47 shows the expression levels of various synTac polypeptides produced in CHO cells.

In FIG. 48 shows the in vivo effect of the synTac polypeptide of the present invention on tumor volume.

In FIG. 49 shows the effect of co-administration of various doses of 4-1BBL/synTac and anti-PD1 antibody on tumor weight and percentage of granzyme B ⁺ tumor infiltrating lymphocytes (TIL).

In FIG. 50A-50B show the amino acid sequences of PD-L1 polypeptides.

In FIG. 51 shows the amino acid sequence of the CD80 polypeptide.

In FIG. 52 shows the amino acid sequence of the ICOS-L polypeptide.

In FIG. 53 shows the amino acid sequence of the OX40L polypeptide.

In FIG. 54 shows the amino acid sequence of the PD-L2 polypeptide.

In FIG. 55 shows the amino acid sequence of the CD86 (B7-2) polypeptide.

In FIG. 56 shows the amino acid sequence of the Fas Ligand (FAS-L) polypeptide.

Definitions.

The terms polynucleotide and nucleic acid, used interchangeably herein, refer to the polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified , unnatural or derivatized nucleotide bases.

The terms peptide, polypeptide, and protein are used interchangeably herein and refer to the polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

A polynucleotide or polypeptide has a certain percentage of sequence identity with another polynucleotide or polypeptide, which means that when aligned, that percentage of bases or amino acids is the same and they are in the same relative positions when the two sequences are compared. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (eg, BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web at sites including ncbi.nlm.nili. gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-10.

The term conservative amino acid substitution refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, the group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids having amide-containing side chains consists of asparagine and glutamine; the group of amino acids having aromatic side chains consists of phenylalanine, tyrosine and tryptophan; the group of amino acids having basic side chains consists of lysine, arginine and histidine; the group of amino acids having acidic side chains consists of and aspartate; and the group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Typical groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinarginine, alanine-valine-glycine and asparagine-glutamine.

As used herein, the term linking (for example, in relation to linking

- 3 042111 multimeric polypeptide that modulates a T cell with a polypeptide (for example, a T cell receptor) on a T cell) refers to a non-covalent interaction between them. Binding interactions are usually characterized by a dissociation constant (K _D ) less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M, less than 10 ^-9 M, less than 10 ^-10 M, less than 10 ^-11 M, less than 10 ^-12 M, less than 10 ^-13 M, less than 10 ^-14 M, or less than 10 ^-15 M. Affinity refers to the strength of binding, increased binding affinity is correlated with lower K _D .

As used herein, the term immunological synapse or immune synapse generally refers to the natural interface between two interacting adaptive immune response immune cells, including, for example, the interface between an antigen presenting cell (APC) or target cell and an effector cell, e.g., a lymphocyte, an effector T cell. natural killer cell and the like. The immunological synapse between the APC and the T cell is usually initiated by the interaction of the T cell antigen recognition receptor and major histocompatibility complex molecules, for example, as described in Bromley et al., Annu. Rev. Immunol. 2001;19:375-96; the contents of which are incorporated herein by reference in their entirety.

A T cell includes all types of CD3-expressing immune cells, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Treg), and NK-T cells.

Costimulatory polypeptide (also referred to herein as immunomodulatory polypeptide), as used herein, includes a polypeptide on an antigen presenting cell (APC) (e.g., dendritic cell, B cell, etc.) that specifically binds a recognized costimulatory polypeptide (also referred to here as a recognized immunomodulatory polypeptide) on the T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, the binding of the TCR/CD3 complex to a major histocompatibility complex (MCHC) polypeptide loaded with a T cell response-mediating peptide including, but not limited to, proliferation, activation, differentiation, and the like. The costimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand ( ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4, HVEM, agonist or antibody that binds a toll-like receptor to a ligand that specifically binds to B7-H3. A costimulatory ligand also includes, among other things, an antibody that specifically binds to a costimulatory molecule present in a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS , lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

The modulating domain (MOD) of a T cell modulating multimeric polypeptide includes a co-stimulatory polypeptide, for example an IL-2 polypeptide, such as a variant IL-2 polypeptide.

In the context of this document, heterologous means a nucleotide or polypeptide that cannot be found in a natural nucleic acid or natural protein, respectively.

As used herein, the term recombinant means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence different from from endogenous nucleic acids that can be found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a range of synthetic oligonucleotides to produce a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in a cell or in a cell-free transcription and translation system.

The terms recombinant expression vector or DNA construct are used interchangeably herein to refer to a DNA molecule containing a vector and a single insert. Recombinant expression vectors are typically generated for the purpose of expressing and/or propagating the insert(s) or for constructing other recombinant nucleotide sequences. Inserts may or may not be operably linked to a promoter sequence and may or may not be operably linked to regulatory DNA sequences.

The terms antibodies and immunoglobulin include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies ( scAb), single domain antibodies (dAb), heavy chain single domain antibodies, light chain single domain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins containing an antigen-binding (herein also referred to as antigen binding) portion of an antibody and a non-antibody protein. In addition, the term encompasses Fab'-, Fv-, F(ab') ₂ - and other antibody fragments that retain specific binding to the antigen.

- 4 042111 nom, as well as monoclonal antibodies. In the context of this document, a monoclonal antibody is an antibody produced by a group of identical cells that are produced in one cell by multiple cellular replication. In other words, a clone of cells produces only one type of antibody. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art (eg, antibodies obtained from antibody phage display libraries) can also be used. The antibody may be monovalent or bivalent. The antibody may be an immunoglobulin (Ig) monomer, which is a Y-shaped molecule composed of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

As used herein, the term humanized antibody refers to an antibody containing portions of antibodies of different origin, at least one portion containing amino acid sequences of human origin. For example, a humanized antibody may comprise parts derived from a non-human immunoglobulin with the desired specificity, such as from a mouse, and derived from human-derived immunoglobulin sequences (eg, a chimeric immunoglobulin), chemically linked together using conventional techniques (eg, synthetically) or obtained as a contiguous polypeptide using genetic engineering techniques (for example, DNA encoding protein portions of a chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized antibody is an antibody containing one or more antibody chains that contain a CDR derived from a non-human antibody and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also covered by the term humanized immunoglobulin. See, for example, Cabilly et al., US patent No. 4816567; Cabilly et al., EP 0125023 B1; Boss et al., US patent No. 4816397; Boss et al., EP 0120694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European patent No. 0194276 B1; Winter, US patent No. 5225539; Winter, European Patent No. 0239400 B1; Padlan, E.A. et al., European Patent Application No. 0519596 A1. See also Ladner et al., US Pat. No. 4,946,778; Huston, US Pat. No. 5,476,786 and Bird, R.E. et al., Science, 242: 423-426 (1988)) regarding single chain antibodies.

For example, humanized antibodies can be made using synthetic and/or recombinant nucleic acids to generate genes (eg, DNA) encoding the desired humanized strand. For example, nucleic acid (e.g., DNA) sequences targeting humanized variable regions can be constructed using NDP-assisted mutagenesis techniques to alter DNA sequences encoding a human or humanized chain, such as a pre-humanized variable region DNA template ( see Kamman, M., et al., Nucl Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-302 (1991)). Through the use of these or other suitable methods, variants can also be easily obtained. For example, cloned variable regions can be mutated and variant-coding sequences with desired specificity can be selected (e.g., from a phage library; see Krebber et al., US Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213 published 1 April 1993)).

Antibody fragments are part of an intact antibody, such as the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fab-, Fab'-, F(ab') ₂ - and Fv-fragments; diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 10571062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Cleavage of antibodies with papain results in two identical antigen-binding fragments, called Fab fragments, each with one antigen-binding site, and a residual Fc fragment, a designation that reflects the ability to easily crystallize. Treatment with pepsin produces an F(ab')2 fragment which has two antigen-binding active sites and is still capable of cross-linking to antigen.

Fv is the minimum antibody fragment that contains a complete antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even one variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab-fragments differ from Fab'-fragments by the addition of several residues

- 5 042111 at the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. As used herein, Fab'-SH is the designation for a Fab' fragment in which the cysteine residues of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments were originally generated as pairs of Fab' fragments that have hinged cysteines between them. Other methods for chemically linking antibody fragments are also known.

Light chain antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further divided into types, for example, IgG2a and IgG2b.

Single chain Fv or sFv or scFv antibody fragments contain the V _H and V _L domains of the antibody, these domains being in the same polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and V _L domains, enabling the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term diabodies refers to small antibody fragments with two antigen binding sites, the fragments of which contain a heavy chain variable domain (V _H ) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Due to the use of a linker that is too short to allow two domains to join in one strand, the domains are forced to combine with complementary domains of the other strand and create two antigen binding sites. Diabodies are described in more detail, for example, in European patent 404,097; WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. sci. USA 90:6444-6448.

In the context of this document, the term affinity refers to the equilibrium constant for the reversible binding of two agents (eg, antibody and antigen) and is expressed as a dissociation constant (KD). Affinity can be at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold , at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more, at least 20 times more, at least 30 times more, according to at least 40 times more, at least 50 times more, at least 60 times more, at least 70 times more, at least 80 times more, at least 90 times more, at least 100 times greater, or at least 1000 times or more greater than the affinity of the antibody for unrelated amino acid sequences. The affinity of an antibody for a target protein may, for example, be from about 100 nanomoles (nM) to about 0.1 nM, from about 100 nM to about 1 picomole (pM), or from about 100 nM to about 1 femtomol (fM) or more. . In the context of this document, the term avidity refers to the resistance of a complex of two or more agents to dissociation after dilution. As used herein, the terms immunoreactive and preferably binds are used interchangeably with respect to antibodies and/or antigen-binding fragments.

The term bonding refers to the direct association of two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic interaction and/or interaction with hydrogen bonds, including interactions such as salt and water bridges. The term specific binding refers to binding with an affinity of at least 10' ⁷ M or more, for example, 5χ 10' ⁷ M, 10' ⁸ M, 5x10'8 M or more. The term non-specific binding refers to binding with an affinity of less than about 10' ⁷ M, for example, binding with an affinity of 10' ⁶ M, 10' ⁵ M, 10' ⁴ M, etc.

As used herein, the term CDR or complementarity determining region is intended to refer to non-contiguous antigen-binding active sites that are within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, Sequences of proteins of immunological interest (1991) (also referred to herein as Kabat 1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where definitions include overlap or subgroups of amino acid residues when compared to each other. However, the use of any of the definitions to refer to the CDR of an antibody, or grafted antibodies, or variants thereof, is encompassed within the scope of said term as defined and used herein. Amino acid residues that span the CDRs as defined in the references cited above are shown in the table below for comparison. CDRs listed in Table. 2 have been determined according to Kabat 1991.

- 6 042111

Table. CDR definitions

Kabat ¹ Chothia ² MacCallum ³ Vh CDR-1 31-35 26-32 30-35 V _H CDR-2 50-65 53-55 47-58 Vh CDR-3 95-102 96-101 93-101 Vl CDR-1 24-34 26-32 30-36 Vl CDR-2 50-56 50-52 46-55 Vl CDR-3 89-97 91-96 89-96

’Numbering of residues according to the nomenclature of Kabat et al. higher.

² Numbering of residues according to the nomenclature of Chothia et al. higher.

³ Numbering of residues according to the nomenclature of MacCallum et al. higher.

As used herein, the terms CDR-L1, CDR-L2, and CDR-L3 refer to the first, second, and third CDRs of the light chain variable region, respectively. As used herein, the terms CDR-H1, CDR-H2, and CDR-H3 refer to the first, second, and third heavy chain variable region CDRs, respectively. As used herein, the terms CDR1, CDR-2, and CDR-3 refer, respectively, to the first, second, and third variable region CDRs of any of the chains.

As used herein, the term framework, when used with respect to the variable region of an antibody, is intended to refer to all amino acid residues outside the CDRs within the variable region of an antibody. The variable region framework is typically a discontinuous amino acid sequence of about 100-120 amino acids in length, but is intended to refer only to those amino acids that are outside the CDR. In the context of this document, the term framework is intended to refer to each framework domain that is separated by a CDR.

The terms treatment, treat, and the like are used herein to denote the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with the disease. As used herein, the term treatment encompasses any treatment of a disease or symptom in a mammal, and includes:

(a) preventing the onset of a disease or symptom in a subject who may be predisposed to developing the disease or symptom but who has not yet been diagnosed with that disease or symptom;

(b) inhibiting the disease or symptom, i. e. cessation of its development; or (c) alleviating the disease, i.e. disease regression.

The therapeutic agent may be administered before, during, or after the onset of a disease or injury. The treatment of the current disease, in which the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirable to carry out until the complete loss of function in the affected tissues. Desirably, the therapy is administered to the subject during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

The terms individual, subject, host, and patient are used interchangeably herein and refer to any mammalian subject requiring diagnosis, treatment, or therapy. Mammals include, for example, humans, non-human primates, rodents (eg, rats; mice), lagomorphs (eg, rabbits), ungulates (eg, cows, sheep, pigs, horses, goats, and the like), etc. .

Before proceeding with the description of the present invention, it should be appreciated that this invention is not limited to the particular embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is used only to describe specific implementations and is not limiting, since the scope of the present invention is limited only by the appended claims.

When a range of values is given, it is to be understood that this invention covers every intermediate value, to the tenth decimal place of the lower limit, unless the context expressly states otherwise, between the upper and lower limits of that range, as well as any other specified or intermediate value within such specified range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in this invention, subject to any limit specifically excluded in the specified range. If the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in this invention.

Unless otherwise noted, all technical and scientific terms used in this document

- 7 042111 have a generally accepted meaning, understandable to any person skilled in the art to which the present invention pertains. While methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials will now be described. All publications mentioned in this document are incorporated herein by reference for the purpose of disclosing and describing the methods and/or materials in connection with which these publications are cited.

It should be noted that the use of the singular herein and in the appended claims includes reference to the plural unless the context clearly indicates otherwise. Thus, for example, reference to an IL-2 polypeptide variant includes a plurality of such polypeptides, and reference to an HLA class I heavy chain polypeptide includes reference to one or more HLA class I heavy chain polypeptides and their equivalents, is known to those skilled in the art. etc. It should also be noted that the claims may be amended to remove any optional elements. Therefore, this statement should serve as a preliminary basis for the use of such exhaustive terminology as exclusively, only, etc. in connection with the indication of the elements of the claims, or the use of a negative feature.

It should be understood that certain features of the present invention, which are described in the context of separate embodiments for clarity, may also be presented in combination as a single embodiment. Conversely, various features of the present invention, which for brevity are described in the context of a single implementation option, can also be presented separately or in any suitable subcombination. All combinations of embodiments that relate to this invention are expressly included in this invention and are described herein in the same way as if all combinations, without exception, were separately and explicitly described. In addition, all subcombinations of the various embodiments and their elements are also expressly included in this invention and are described herein in the same way as if all subcombinations without exception were separately and explicitly described.

The publications discussed herein are presented solely in relation to their content prior to the filing date of this application. Nothing herein should be interpreted as an admission that this invention is not entitled to predate such publication by virtue of an earlier invention. Moreover, the dates of the submitted publications may differ from the actual dates of the publications, which requires independent confirmation.

Detailed Description of the Invention

The present invention provides methods of treatment comprising administering to an individual in need thereof a multimeric T cell modulating polypeptide (synTac multimeric polypeptide) and at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent is an immune checkpoint inhibitor. In some cases, an immune checkpoint inhibitor is an antibody specific for an immune checkpoint. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The present invention relates to methods comprising administering a multimeric polypeptide (synTac) to an individual who is being treated with an immune checkpoint inhibitor.

A T-cell modulating multimeric polypeptide is also referred to herein as a synTac polypeptide or synTac multimeric polypeptide or simply synTac. The synTac polypeptide contains a modulating domain. In some cases, the modulating domain contains a wild-type amino acid sequence, such as an amino acid sequence found in a naturally occurring modulating polypeptide. In some instances, the modulating domain is a variant of the modulating domain, wherein the variant of the modulating domain exhibits reduced binding affinity for the immunomodulatory polypeptide compared to the affinity of the wild-type modulatory domain for the immunomodulatory polypeptide. The synTac polypeptide can modulate the activity of a target T cell. A synTac polypeptide containing a variant modulatory domain provides increased specificity for target cells.

In some instances, the method of treatment of the present invention comprises administering to an individual in need thereof synTac and an immune checkpoint inhibitor. In some cases, synTac and an immune checkpoint inhibitor provide a synergistic effect compared to the effect of synTac when administered alone (monotherapy) or the immune checkpoint inhibitor when administered alone (monotherapy).

The combination of synTac and an immune checkpoint inhibitor is in some cases more effective than the additive effects of synTac given as monotherapy or an immune checkpoint inhibitor given as monotherapy. For example, in some cases, the synergistic effect of synTac and an immune checkpoint inhibitor allows the use of lower doses of synTac or an immune checkpoint inhibitor and/or less frequent administration of syn

- 8 042111

Tac or an immune checkpoint inhibitor to an individual in need thereof. The ability to use lower doses of therapeutic agents (synTac inhibitor or immune checkpoint) and/or administer such agents less frequently may reduce the toxicity or other adverse side effects that may be associated with administering a therapeutic agent in monotherapy without compromising the efficacy of the therapeutic agent in treatment. In addition, the synergistic effect of synTac and an immune checkpoint inhibitor may result in increased clinical benefit compared to the clinical benefit obtained with synTac monotherapy or immune checkpoint inhibitor monotherapy. Examples of clinical benefit include, for example, reduction in tumor mass in an individual; reduction in the number of cancer cells in humans; increased human survival time; increased remission time; etc. Finally, the synergistic effect of synTac and an immune checkpoint inhibitor may reduce adverse or unwanted side effects associated with synTac monotherapy or immune checkpoint inhibitor monotherapy.

Immune checkpoint inhibitors.

Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD- L2. In some instances, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is a checkpoint inhibitory molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA .

In some cases, an immune checkpoint inhibitor is an antibody specific for an immune checkpoint. In some cases, the immune checkpoint antibody is a monoclonal antibody. In some cases, the immune checkpoint antibody is humanized or deimmunized such that the antibody does not substantially elicit an immune response in the individual. In some cases, the antibody is a humanized monoclonal antibody. In some cases, the immune checkpoint antibody is a deimmunized monoclonal antibody. In some cases, the immune checkpoint antibody is a fully human monoclonal antibody. In some cases, the immune checkpoint antibody inhibits the binding of the immune checkpoint polypeptide to the ligand of the immune checkpoint polypeptide. In some instances, the immune checkpoint antibody inhibits the binding of the immune checkpoint polypeptide to the immune checkpoint polypeptide receptor.

Antibodies, such as monoclonal antibodies, which are specific for immune checkpoints and which act as immune checkpoint inhibitors, are known in the art. See, for example, Wurz et al. (2016) Ther. Adv. Med. oncol. 8:4; and Naidoo et al. (2015) Ann. oncol. 26:2375.

Suitable immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex , Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI- 6469 (Medlmmune/AZ), CP-870, 893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), indoxymod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof.

Suitable anti-LAG3 antibodies include, for example, BMS-986016 and LAG525. Suitable anti-GITR antibodies include, for example, TRX518, MK-4166, INCAGN01876 and MK-1248. Suitable anti-OX40 antibodies include, for example, MEDI0562, INCAGN01949, GSK2831781, GSK-3174998, MOXR-0916, PF04518600 and LAG525. Suitable antibodies to VISTA are presented, for example, in WO 2015/097536.

A suitable dosage of the anti-immune control antibody is from about 1 mg/kg to about 2400 mg/kg per day, for example from about 1 mg/kg to about 1200 mg/kg per day, including from about 50 mg/kg to about 1200 mg /kg per day. Other typical dosages of such agents include about 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg. kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg kg, 2200 mg/kg and 2300 mg/kg per day. An effective dose of an antibody may be administered as two, three, four, five, six, or more divided doses administered separately at appropriate intervals of

- 9 042111 during the day.

Antibodies to PD-1.

In some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody.

Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001 and AMP-224. In some instances, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001. Suitable antibodies to PD1 are described in US Patent Publication No. 2017/0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18.

In some cases, the anti-PD1 antibody is pembrolizumab. Amino acid sequence of pembrolizumab heavy chain:

OVOLVOSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGOGLEWMGGI

NPSNGGTNNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDY

WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQ S SGL YSLS S VVT VP S S SLGTKT YTCNVDHKP SNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:51). The amino acid sequence of the heavy chain variable region (VH) is underlined. Pembrolizumab light chain amino acid sequence: EIVLTOSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYOOKPGQAPRLLIYLAS

YLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO :52). The amino acid sequence of the light chain variable region (VL) is underlined. In some cases, the anti-PD-1 antibody contains the VH and VL regions of pembrolizumab. In some cases, the anti-PD-1 antibody contains the heavy chain and light chain CDRs of pembrolizumab.

In some cases, the anti-PD-1 antibody is nivolumab (also known as MDX1106 or BMS-936558; see e.g. Topalian et al. (2012) N. Eng. J. Med. 366:2443-2454; and US Pat. No. 8,008,449). The amino acid sequence of the heavy chain of nivolumab is:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIW YDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQS SGLYSLS SVVTVPS S SLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:53).

The amino acid sequence of the light chain of nivolumab is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKSSVYACEVQ5.

In some cases, the anti-PD-1 antibody contains the heavy chain and light chain CDRs of nivolumab. Antibodies to CTLA4.

In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, for example, Ribas et al. (2013) J. Clin. oncol. 31:616-22.

In some cases, the anti-CTLA-4 antibody is ipilimumab. The amino acid sequence of the heavy chain of ipilimumab is:

- 10 042111

OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISY DGNNKYYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAIYYCARTGWLGPFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55).

The amino acid sequence of the VH region is underlined.

Amino acid sequence of the light chain of ipilimumab:

EIVLTOSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGQAPRLLIYGAFSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCOOYGSSPWTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSL SSTLTLSKADYEKHKVYACENSFTHQGL5. The amino acid sequence of the VL region is underlined.

In some cases, the anti-CTLA4 antibody contains the VH and VL regions of ipilimumab. In some cases, the anti-CTLA4 antibody contains the heavy chain and light chain CDRs of ipilimumab.

Antibodies to PD-L1.

In some cases, the immune checkpoint inhibitor is an anti-PD-L1 monoclonal antibody. In some instances, the anti-PD-L1 monoclonal antibody is BMS935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some cases, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab see, for example, WO 2011/066389. For atezolizumab, see, for example, US Pat. No. 8,217,149.

In some cases, the anti-PD-L1 antibody is atezolizumab. The amino acid sequence of the heavy chain of atezolizumab is:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPY GGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF Р AVLQ S SGL YSLS S VVT VP S S SLGTQT YICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57).

The amino acid sequence of the light chain of atezolizumab is:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLECSKADYEKHKVY5.

In some embodiments, the anti-PD-L1 antibody comprises the heavy chain and light chain CDRs of atezolizumab.

In some instances, the anti-PDL1 antibody is KN035, a fully humanized anti-PD-L1 single domain antibody fused to a human IgG1 Fc polypeptide. Zhang et al. (2017) Cell Discov. 3:17004; and WO 2017/020801. The single domain portion of the KN035 antibody may contain the amino acid sequence:

OVOLOESGGGLVOPGGSLRLSCAASGKMSSRRCMAWFROAPGKERERVAKLLTTSGST

YLADSVKGRFTISONNAKSTVYLOMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQ

YWGQGTQVTVS (SEQ ID NO:216), where the underlined amino acids are CDR1, CDR2 and CDR3.

Modulating T-cells multimeric polypeptides (synTac).

Multimeric (eg, heterodimeric, heterotrimeric) polypeptides suitable for use in the method of the present invention are described below. Multimeric polypeptides are polypeptides that modulate T cells and are also referred to herein as T cell modulating multimeric polypeptides or synTac (from immunological synapse for T cell

- 11 042111 activation - immunological synapse for T-cell activation).

The T-cell modulating multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory (MOD) domains, wherein the one or more immunomodulatory domains are:

A) at the C-terminus of the first polypeptide;

C) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide. In some cases, the T cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the T-cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, from the N-terminus to the C-terminus:

i) an immunomodulatory domain;

iii) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

In some cases, the T-cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory (MOD) domain.

In some cases, the T-cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) an immunomodulatory domain.

In some cases, the T-cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, from the N-terminus to the C-terminus:

i) an immunomodulatory domain; and ii) a second MHC polypeptide.

In some cases, the T-cell multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide.

In some instances, the multimeric polypeptide contains a non-Ig backbone.

For example, in some instances, the non-Ig scaffold is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or

- 12 042111 silk-like elastin-like polypeptide.

In some instances, the first MHC polypeptide is a β2 microglobulin (v2M) polypeptide; and the second MHC polypeptide is an MHC class I heavy chain polypeptide. 99% or 100% amino acid sequence identity with the amino acid sequence of the b2M polypeptide depicted in FIG. 6. In some instances, the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain. In some cases, the MHC class I heavy chain polypeptide contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity relative to the amino acid sequence shown in one of FIG. 5A-5C. In some instances, the first MHC polypeptide is an MHC class II alpha chain polypeptide; and the second MHC polypeptide is an MHC class II beta chain polypeptide.

An epitope present in a multimeric polypeptide may be a T cell epitope.

In some instances, the multimeric polypeptide comprises an Ig Fc polypeptide. In some instances, an Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, an Ig Fc polypeptide contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity relative to the amino acid sequence, shown in FIG. 4A-4C.

The first polypeptide and the second polypeptide of the multimeric polypeptide may be non-covalently linked. The first polypeptide and the second polypeptide of the multimeric polypeptide may be covalently linked. The first polypeptide and the second polypeptide of the multimeric polypeptide may be covalently linked, the covalent bond being through a disulfide bond. In some cases, the first MHC polypeptide or linker between the epitope and the first MHC polypeptide contains an amino acid substitution to obtain a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to obtain a second Cys residue, and where the disulfide bond is between the first and second Cys residues.

The multimeric polypeptide may include a linker between one or more of: the epitope and the first MHC polypeptide; two copies of the immunomodulatory (MOD) polypeptide; an immunomodulatory polypeptide and a second MHC polypeptide; and a second MHC polypeptide and an Ig Fc polypeptide.

Suitable immunomodulatory polypeptides for inclusion in a T cell multimeric polypeptide include, but are not limited to, 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, IL-2 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

A multimeric polypeptide may include 2 or more immunomodulatory polypeptides. The multimeric polypeptide may include 2 immunomodulatory polypeptides. In some cases, 2 immunomodulatory polypeptides are in tandem. The multimeric polypeptide may include 3 immunomodulatory polypeptides. In some cases, the 3 immunomodulatory polypeptides are in tandem.

The multimeric polypeptide may comprise a third polypeptide, wherein the third polypeptide comprises an immunomodulatory polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the immunomodulatory polypeptide of the first polypeptide or the second polypeptide. In some cases, the third polypeptide is covalently linked to the first polypeptide.

Examples of suitable multimeric polypeptides are described in WO 2017/151940; WO 2017/201210; and PCT/US2017/067663. Disclosures WO 2017/151940, WO 2017/201210 and PCT/US2017/067663 are incorporated here by reference.

MHC polypeptides.

As noted above, the multimeric polypeptide of the present invention includes MHC polypeptides. For purposes of the present invention, the term major histocompatibility complex (MHC) polypeptides includes MHC polypeptides of various species, including human MHC polypeptides (also referred to as human leukocyte antigens (HLA)), rodent (e.g., mice, rats, etc.) MHC polypeptides. and MHC polypeptides from other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., horses, bovines, sheep, goats, etc.), etc.) The term MHC polypeptide is intended to include MHC class I polypeptides (e.g., β-2-microglobulin and MHC class I heavy chain) and MHC class II polypeptides (e.g.

- 13 042111 mer, MHC class II α-polypeptide and MHC class II β-polypeptide).

As noted above, in some embodiments of the multimeric polypeptide of the present invention, the first and second MHC polypeptides are class I MHC polypeptides; for example, in some instances, the first MHC polypeptide is an MHC class I β2 microglobulin (f2M) polypeptide and the second MHC polypeptide is a heavy chain of MHC class I (H-chain). In other cases, the first and second MHC polypeptides are class II MHC polypeptides; for example, in some instances, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other instances, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide.

In some instances, the MHC polypeptide of the multimeric polypeptide of the present invention is a human MHC polypeptide, where human MHC polypeptides are also referred to as human leukocyte antigen (HLA) polypeptides. In some instances, the MHC polypeptide of the multimeric polypeptide of the present invention is an HLA class I polypeptide, such as a β2-microglobulin polypeptide or an HLA class I heavy chain polypeptide. HLA class I heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides , HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides. In some instances, the MHC polypeptide of the multimeric polypeptide of the present invention is an HLA class II polypeptide, such as a class II α chain HLA or a class II β chain HLA. MHC class II polypeptides include MHC class II α and β DP polypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides.

In some instances, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence depicted in one of FIGS. 5A-5C.

HLA-A.

As an example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-A heavy chain amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWARYVVPSGQE (LVETRPAGDGTFQKWARYVVPSGQE

NO:59).

HLA-A (Y84A; A236C).

In some cases, the MHC class I heavy chain polypeptide contains substitutions Y84A and A236C. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-A heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETKVKAHSQTHRVDLGTLRG

C

GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 50) wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with the Cys-12 variant of the β2M polypeptide containing the R12C substitution.

HLA-A (Y84C; A139C).

In some instances, the MHC class I heavy chain polypeptide contains Y84C and A139C substitutions.

- 14 042111

For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-A heavy chain amino acid sequence (Y84C; A139C):

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO: 196), where amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-A A11 (HLA-A11).

As one non-limiting example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-A A11 heavy chain amino acid sequence (also referred to as HLA-A11):

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLLPKPLTLRWE (SEQ ID NO: 197).

Such heavy chain MHC class I can be seen in Asian populations, including populations of people of Asian descent.

HLA-A A11 (Y84A; A236C).

As one non-limiting example, in some cases the MHC class I heavy chain polypeptide is the HLA-A A11 allele, which contains the substitutions Y84A and A236C. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following amino acid sequence of the human HLA-A A11 heavy chain (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGAYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLLPKPLTLRWE (SEQ ID NO: 198) wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the b2M polypeptide variant containing the R12C substitution.

HLA-B.

As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the human HLA-B heavy chain amino acid sequence of:

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY

WDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY

(SEQ ID NO: 199).

HLA-B (Y84A; A236C).

As one non-limiting example, in some cases a heavy chain polypeptide

- 15 042111

Class I MHC is an HLA-B polypeptide that contains Y84A and A236C substitutions. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-B heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY WDRNTQIYKAQAQTDRESLRNLRGAYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY

AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 200) wherein amino acid 84 is Cy3 2 Ala and amino acid 84 is Cy3 2 Ala. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the b2M polypeptide variant containing the R12C substitution.

HLA-B (Y84C; A139C).

In some instances, the MHC class I heavy chain polypeptide contains Y84C and A139C substitutions. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-B heavy chain amino acid sequence (Y84C; A139C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY WDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTCAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:201), где аминокислота 84 представляет собой Cys и аминокислота 139 представляет собой Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-C.

As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the human HLA-C heavy chain amino acid sequence of:

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY

WDRETQNYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 202).

HLA-C (Y84A; A236C).

As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-C polypeptide that contains the Y84A and A236C substitutions. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-C heavy chain amino acid sequence (Y84A; A236C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQNYKRQAQADRVSLRNLRGAYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:203), где аминокислота 84 представляет собой Ala и аминокислота 236 представляет собой Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the b2M polypeptide variant containing the R12C substitution.

HLA-C (Y84C; A139C).

- 16 042111

In some instances, the MHC class I heavy chain polypeptide contains Y84C and A139C substitutions. For example, in some cases, an MHC class I heavy chain polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, at least 99% or 100% amino acid sequence identity with the following human HLA-C heavy chain amino acid sequence (Y84C; A139C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY

WDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTCAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:204), где аминокислота 84 представляет собой Cys и аминокислота 139 представляет собой Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

In some instances, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence depicted in one of FIGS. 3A-3C.

As an example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 25365 of the human HLA-A heavy chain polypeptide amino acid sequence shown in FIG. 3A.

As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 25-362 of the human HLA-B heavy chain polypeptide sequence of FIG. 3B.

As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 25-362 of the human HLA-C heavy chain polypeptide of FIG. 3C.

As another example, an MHC class I heavy chain polypeptide of a multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence:

GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQ

EGPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLR GYQQFAYDGRDYIALNEDLKTWTAADTAALITRRKWEQAGDAEYYRAYLEGECVEWL RRYLELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQ DMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRW (SEQ ID NO:60).

The b2-microglobulin (b2M) polypeptide of the multimeric polypeptide can be a human b2M polypeptide, a non-human primate b2M polypeptide, a mouse 32M polypeptide, and the like. In some cases, the b2M polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99 % or 100% amino acid sequence identity with the b2M amino acid sequence depicted in FIG. 6. In some cases, the b2M polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least at least 99% or 100% amino acid sequence identity with amino acids 21 to 119 of the b2M amino acid sequence depicted in FIG. 6.

- 17 042111

In some instances, the MHC polypeptide contains a single amino acid substitution relative to the reference MHC polypeptide (wherein the reference MHC polypeptide may be a wild-type MHC polypeptide), where the single amino acid substitution replaces the amino acid with a cysteine (Cys) residue. Such cysteine residues, when present in the MHC polypeptide of the first polypeptide of the multimeric polypeptide of the present invention, can form a disulfide bond with a cysteine residue present in the second polypeptide chain of the multimeric polypeptide of the present invention.

In some instances, the first MHC polypeptide in the first MHC polypeptide and/or the second MHC polypeptide in the second MHC polypeptide comprises an amino acid substitution to replace the amino acid with cysteine, wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a cysteine in the second MHC polypeptide, where the cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide or where the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide.

For example, in some cases, one of the following pairs of residues in HLA P2 microglobulin and HLA class I heavy chain is replaced by cysteines (where the residue numbers correspond to those of the mature polypeptide):

1) residue v2M 12, heavy chain residue HLA class I 236;

2) residue v2M 12, heavy chain residue HLA class I 237;

3) residue v2M 8, heavy chain residue HLA class I 234;

4) residue v2M 10, heavy chain residue HLA class I 235;

5) residue v2M 24, heavy chain residue HLA class I 236;

6) residue v2M 28, heavy chain residue HLA class I 232;

7) residue v2M 98, heavy chain residue HLA class I 192;

8) residue v2M 99, heavy chain residue HLA class I 234;

9) residue v2M 3, heavy chain residue HLA class I 120;

10) residue v2M 31, heavy chain residue HLA class I 96;

11) residue v2M 53, heavy chain residue HLA class I 35;

12) residue v2M 60, heavy chain residue HLA class I 96;

13) residue v2M 60, heavy chain residue HLA class I 122;

14) residue v2M 63, heavy chain residue HLA class I 27;

15) b2M Arg3 residue, heavy chain residue of HLA class I Gly120;

16) His31 b2M residue, HLA class I heavy chain residue Gln96;

17) Asp53 b2M residue, HLA class I Arg35 heavy chain residue;

18) B2M Trp60 residue, heavy chain residue of HLA class I Gln96;

19) B2M Trp60 residue, heavy chain residue of HLA class I Asp122; 20) B2M Trp63 residue, heavy chain residue of HLA class I Tyr27;

21) B2M Lys6 residue, heavy chain residue of HLA class I Glu232;

22) B2M Gln8 residue, heavy chain residue of HLA class I Arg234;

23) B2M Tyr10 residue, HLA class I Pro235 heavy chain residue; 24) B2M Ser11 residue, heavy chain residue of HLA class I Gln242;

25) b2M residue Asn24, HLA class I heavy chain residue Ala236;

26) b2M Ser28 residue, heavy chain residue of HLA class I Glu232;

27) Asp98 b2M residue, HLA class I His192 heavy chain residue; And

28) b2M Met99 residue, heavy chain residue of HLA class I Arg234.

The MHC/HLA class I heavy chain amino acid numbering refers to the mature MHC/HLA class I heavy chain without the signal peptide. For example, in the amino acid sequence depicted in FIG. 5A, which includes the signal peptide, Gly120 is Gly144; Gln96 is Glnl20; etc. In some cases, the b2M polypeptide contains an R12C substitution and the HLA class I heavy chain contains an A236C substitution; in such cases, a disulfide bond is formed between Cys-12 of the b2M polypeptide and Cys-236 of the HLA class I heavy chain. Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence (ie, residue 260 of the amino acid sequence depicted in FIG. 5B) is replaced by Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence (ie, residue 260 of the amino acid sequence depicted in FIG. 5C) is replaced by Cys. In some cases, residue 32 (corresponding to mature b2M Arg-12) of the amino acid sequence depicted in FIG. 6 is substituted by Cys.

In some cases, the b2M polypeptide contains the amino acid sequence:

- 18 042111

IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:61).

In some cases, the β2M polypeptide contains the amino acid sequence: IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48).

In some cases, an HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ IDNO:59).

In some cases, an HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:62).

In some cases, an HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:50).

In some cases, the b2M polypeptide contains the following amino acid sequence: IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:48);

and the HLA class I heavy chain polypeptide of the multimeric polypeptide of the present invention contains the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO :62), где остатки Cys, которые подчеркнуты и выделены жирным шрифтом, образуют дисульфидную связь друг с другом в мультимерном полипептиде.

In some cases, the b2M polypeptide contains the amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:48).

framework polypeptides.

The T cell modulating multimeric polypeptide comprises an Fc polypeptide or other suitable backbone polypeptide.

Suitable framework polypeptides include antibody frameworks and non-antibody frameworks. Non-antibody scaffolds include e.g. albumin, polypep

- 19 042111 XTEN tide (extended recombinant), transferrin, Fc receptor polypeptide, elastin-like polypeptide (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g. -Gly-X-Gly; SEQ ID NO: 212) where X is any amino acid other than proline), albumin-binding polypeptide, silk-like polypeptide (see e.g. Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165) silk-like elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075) and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682 and US 2009/0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

Suitable scaffold polypeptides, in some cases, are polypeptides that increase the half-life. Thus, in some cases, a suitable framework polypeptide increases the in vivo half-life (eg, serum half-life) of a multimeric polypeptide compared to a control multimeric polypeptide lacking a framework polypeptide. For example, in some cases, a framework polypeptide increases the in vivo half-life (e.g., serum half-life) of a multimeric polypeptide compared to a control multimeric polypeptide lacking a framework polypeptide by at least about 10%, with at least about 15 %, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times. For example, in some instances, an Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of a multimeric polypeptide compared to a control multimeric polypeptide lacking an Fc polypeptide by at least about 10%, at least about 15%. , at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times , at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times.

Fc polypeptides.

In some instances, the first and/or second polypeptide chain of a multimeric polypeptide comprises an Fc polypeptide. The Fc polypeptide of the multimeric polypeptide may be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, at least about 98%, at least about 99%, or 100% amino acid sequence identity relative to the amino acid sequence of the Fc region depicted in FIG. 4A-C. In some cases, the Fc region contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% amino acid sequence identity with the human IgG1 Fc polypeptide depicted in FIG. 4A. In some cases, the Fc region contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% amino acid sequence identity with the human IgG1 Fc polypeptide shown in FIG. 4A; and contains the replacement N77; for example, the Fc polypeptide contains the N77A substitution. In some cases, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% amino acid sequence identity with the human IgG2 Fc polypeptide depicted in FIG. 4A; for example, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% amino acid sequence identity with amino acids 99-325 of the human IgG2 Fc polypeptide depicted in FIG. 4A. In some cases, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% identity

- 20 042111 amino acid sequence of the human IgG3 Fc polypeptide shown in FIG. 4A; for example, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% amino acid sequence identity with amino acids 19-246 of the human IgG3 Fc polypeptide depicted in FIG. 4A. In some cases, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% amino acid sequence identity with the human IgM Fc polypeptide shown in FIG. 4B; for example, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% amino acid sequence identity with amino acids 1-276 of the human IgM Fc polypeptide shown in FIG. 4B. In some cases, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, with at least about 98%, at least about 99%, or 100% amino acid sequence identity with the human IgA Fc polypeptide shown in FIG. 4C; for example, the Fc polypeptide contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% amino acid sequence identity with amino acids 1-234 of the human IgA Fc polypeptide shown in FIG. 4C.

In some instances, the Fc polypeptide present in the multimeric polypeptide contains the amino acid sequence depicted in FIG. 33A (Fc human IgG1). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of N297 with an amino acid other than asparagine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33C (human IgG1 Fc containing N297A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of L234 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of L235 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33D (human IgG1 Fc containing L234A substitution and L235A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of P331 with an amino acid other than proline; in some cases, the replacement is a P331S replacement. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for substitutions at L234 and L235 with amino acids other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the substitutions in L234 and L235 with non-leucine amino acids and the substitution of P331 with non-proline amino acids. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure contains the amino acid sequence depicted in FIG. 33B (human IgG1 Fc containing substitutions L234F, L235E and P331S). In some instances, the Fc polypeptide present in the multimeric polypeptide is an IgG1 Fc polypeptide that contains the L234A and L235A substitutions.

Linkers.

The multimeric polypeptide may include linker peptides located, for example, between the epitope and the MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between the first immunomodulatory polypeptide and the second immunomodulatory polypeptide; or between a second immunomodulatory polypeptide and a third immunomodulatory polypeptide.

For example, a multimeric polypeptide may include linker peptides located on

- 21 042111 example, between an epitope and an MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between the first version of the IL-2 polypeptide and the second version of the IL-2 polypeptide; or between a second IL-2 polypeptide variant and a third IL-2 polypeptide variant. As another example, a multimeric polypeptide may include linker peptides located, for example, between an epitope and an MHC polypeptide; between an MHC polypeptide and an immunomodulatory polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between the first version of the 4-1BBL polypeptide and the second version of the 4-1BBL polypeptide; or between a second variant of the 4-1BBL polypeptide and a third variant of the 4-1BBL polypeptide.

Suitable linkers (also referred to as spacers) can be readily selected and can be any of a number of suitable lengths such as 1 amino acid to 25 amino acids, 3 amino acids to 20 amino acids, 2 amino acids to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. A suitable linker may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids long.

Examples of linkers include glycine (G)n polymers, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 210) and (GGGS)n (SEQ ID NO: 211), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers can be used, Gly and Ser are both relatively unstructured so can serve as a neutral bond between the components. Polymers of glycine can be used, glycine gains significantly more phi-psi space than even alanine and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142(1992)).

Example linkers may contain amino acid sequences including, but not limited to,

GGSG (SEQ ID NO:65), GGSGG (SEQ ID NO:66),

GSGSG (SEQ ID NO:67), GSGGG (SEQ ID NO:68), GGGSG (SEQ ID NO:69), GSSSG (SEQ

ID NO :70) and the like. Examples of linkers may include, for example, Gly (Ser ₄ ) _n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some cases, the linker contains the amino acid sequence (GSSSS) _n (SEQ ID NO: 71), where n is 4. In some cases, the linker contains the amino acid sequence (GSSSS) n (SEQ ID NO: 72), where n is 5. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO : 205), where n is 1. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 206), where n is 2. In some cases, the linker contains the amino acid sequence (GGGGS) n (SEQ ID NO: 207 ), where n is 3. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 208), where n is 4. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 209), where n is 5. In some cases, the linker contains the amino acid sequence AAAGG (SEQ ID NO: 73).

In some cases, the linker polypeptide present in the first polypeptide of the multimeric polypeptide of the present invention includes a cysteine residue that can form a disulfide bond with a cysteine residue present in the second polypeptide of the multimeric polypeptide of the present invention. In some cases, for example, a suitable linker contains the amino acid sequence

GCGASGGGGSGGGGS (SEQ ID NO:74).

Epitopes.

An epitope (peptide presenting one or more epitopes) present in a multimeric polypeptide of the present invention may be from about 4 amino acids to about 25 amino acids in length, for example, an epitope may be from 4 amino acids (aa) to 10 aa, from 10 to 15 ak, from 15 to 20 ak or from 20 to 25 ak. For example, an epitope present in a multimeric polypeptide of the present invention may be 4 amino acids (aa), 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14 ak, 15 ak, 16 ak, 17 ak, 18 ak, 19 ak, 20 ak, 21 ak, 22 ak, 23 ak, 24 ak or 25 ak. In some instances, an epitope present in a multimeric polypeptide of the present invention is 5 amino acids to 10 amino acids in length, such as 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

The epitope present in the multimeric polypeptide of the present invention is specifically associated with a T cell, that is, the epitope is specifically associated with an epitope-specific T cell. An epitope-specific T cell binds an epitope having a reference amino acid sequence but does not substantially bind an epitope that differs from the reference amino acid sequence. For example, an epitope-specific T cell binds an epitope having a reference amino acid sequence and binds an epitope that differs from the reference amino acid sequence, if at all, with an affinity of less than 10 ^-6 M, less than 10 ^-5 M, or

- 22 042111 less than 10 ^-4 M. An epitope-specific T cell can bind the epitope for which it is specific with an affinity of at least 10 ^-7 M, at least 10 ^-8 M, at least 10 ^-9 M or at least

10 ^-10 M.

Suitable epitopes include, but are not limited to, those present on an antigen associated with cancer. Cancer-associated antigens include, but are not limited to, α-folate receptor; carbonic anhydrase IX (CAiX); CD19; CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor; ganglioside antigen GD2; HER2/Neu; IL-13R-a2; kappa light chain; LeY; cell adhesion molecule L1; melanoma-associated antigen (MAGE); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); tumor-associated glycoprotein-72 (TAG-72); and vascular endothelial growth factor receptor-2 (VEGF-R2). See, for example, Vigneron et al. (2013) Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int'l Article ID 948501. In some cases, the epitope is an epitope of the E7 antigen of the human papillomavirus; see, for example, Ramos et al. (2013) J. Immunother. 36:66.

In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO: 75). In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO: 76). In some cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO: 77). In some cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO: 78). See, for example, Ressing et al. ((1995) J. Immunol. 154:5934) for additional suitable HPV epitopes.

immunomodulatory polypeptides.

Suitable immunomodulatory polypeptides include, but are not limited to, IL-2 polypeptide, 4-1BBL polypeptide, B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the PD-L1 polypeptide depicted in FIG. 50A or FIG. 50V.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the CD80 polypeptide shown in FIG. 51.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the ICOS-L polypeptide depicted in FIG. 51.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the OX40L polypeptide depicted in FIG. 53.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the PD-L2 polypeptide depicted in FIG. 54.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the CD86 polypeptide shown in FIG. 55.

In some instances, the immunomodulatory polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid identity. sequences with the amino acid sequence of the FAS-L polypeptide shown in FIG. 56.

In some instances, an immunomodulatory polypeptide present in synTac exhibits reduced binding affinity for a recognized co-immunomodulatory polypeptide expressed on the surface of a T cell compared to the binding affinity of a wild-type immunomodulatory polypeptide for the same recognized co-immunomodulatory polypeptide. In some cases, when the synTac contains an immunomodulatory polypeptide with reduced affinity, the synTac polypeptide exhibits reduced binding to the recognized immunomodulatory polypeptide expressed on the surface of the T cell. For example, in some instances, a synTac polypeptide that contains an immunomodulatory polypeptide with reduced affinity binds a recognized co-immunomodulatory polypeptide with a binding affinity of

- 23 042111 at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, and at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less binding affinity of a control synTac polypeptide containing a wild type immunomodulatory polypeptide to same related immunomodulatory polypeptide.

Determination of binding affinity.

The binding affinity between an immunomodulatory polypeptide and its recognized co-immunomodulatory polypeptide can be determined by biolayer interferometry (BLI) using a purified immunomodulatory polypeptide and a purified recognized co-immunomodulatory polypeptide. The binding affinity between the synTac of the present invention and its recognized co-immunomodulatory polypeptide can also be determined by BLI using a purified synTac and a recognized co-immunomodulatory polypeptide. BLI methods are well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. screen. 20(4):498-507; and Shah and Duncan (2014) J. Vis. Exp. 18:e51383. The specific and relative binding affinities described in this disclosure between an immunomodulatory polypeptide and its recognized immunomodulatory polypeptide or between synTac and its recognized immunomodulatory polypeptide can be determined using the following procedures.

To determine the binding affinity between the synTac of the present invention and its recognized co-immunomodulatory polypeptide, a BLI assay can be performed using an Octet RED 96 instrument (Pal ForteBio) or a similar instrument, as indicated below. To determine the binding affinity of a T cell modulating multimeric polypeptide (e.g., synTac of the present invention or a control T cell modulating multimeric polypeptide (wherein the control T cell modulating multimeric polypeptide contains a wild type immunomodulatory polypeptide)), the T cell modulating multimeric polypeptide is immobilized on insoluble carrier (biosensor). The immobilized T-cell modulating multimeric polypeptide is the target. Immobilization can be accomplished by immobilizing the capture antibody on an insoluble carrier, wherein the capture antibody immobilizes a multimeric T-cell modulating polypeptide. For example, immobilization can be accomplished by immobilizing anti-Fc antibodies (e.g., anti-human IgG Fc) on an insoluble carrier, where the immobilized anti-Fc antibody binds and immobilizes a T cell modulating multimeric polypeptide (wherein the T cell modulating multimeric polypeptide comprises an Ig Fc polypeptide) . The co-immunomodulatory polypeptide is applied at several different concentrations to the immobilized multimeric T-cell modulating polypeptide and the response of the instrument is recorded. Assays are performed in liquid medium containing 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% non-ionic detergent Tween 20. modulating T-cells multimeric polypeptide is carried out at 30°C. An anti-MHC class I monoclonal antibody can be used as a positive control for binding affinity. . 123:342) with K _D 7 nM. A standard curve can be generated using serial dilutions of an anti-MHC class I monoclonal antibody. An anti-MHC class I co-immunomodulatory polypeptide or mAb is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces:

i) from the immobilized polypeptide (target); and ii) an internal reference layer.

Changing the number of molecules (analyte; eg, co-immunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a change in the interference pattern; this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of the interaction between the target and the analyte are the association constant (k _a ) and the dissociation constant (kd). The ratio of these two values (kd/ _a ) results in the affinity constant Kd.

As noted above, determination of binding affinity between an immunomodulatory polypeptide (eg, IL-2 or IL-2 variant) and its recognized co-immunomodulatory polypeptide (eg, IL-2R) can also be determined using BLI. The analysis is similar to that described above for the multimeric synTac polypeptide. BLI analysis can be performed using an Octet RED 96 instrument (Pal ForteBio) or a similar instrument as follows. A component of the immunomodulatory synTac polypeptide of the present invention (eg, a variant of the IL-2 polypeptide of the present invention); and a control immunomodulatory polypeptide (wherein the control immuno

- 24 042111 nomodulatory polypeptide contains a wild-type immunomodulatory polypeptide, for example, wild-type IL2) immobilized on an insoluble carrier (biosensor). The immunomodulatory polypeptide is the target. Immobilization can be accomplished by immobilizing the capture antibody on an insoluble carrier, wherein the capture antibody immobilizes the immunomodulatory polypeptide. For example, if the target is fused with an immunoaffinity tag (e.g., FLAG, human IgG Fc), immobilization can be accomplished by immobilization with an appropriate immunoaffinity tagged antibody (e.g., human IgG anti-Fc) on an insoluble carrier, where the immobilized antibodies are bound and immobilized an immunomodulatory polypeptide (wherein the immunomodulatory polypeptide comprises an Ig Fc polypeptide). The co-immunomodulatory polypeptide (or polypeptides) is applied at several different concentrations to the immobilized immunomodulatory polypeptide and the response of the instrument is recorded. Alternatively, a co-immunomodulatory polypeptide (or polypeptides) is immobilized in a biosensor (e.g., for an IL-2 receptor heterotrimer, as a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer) and the immunomodulatory polypeptide is applied at several different concentrations to the immobilized co-immunomodulatory polypeptide and the response of the instrument is recorded. Assays are performed in liquid medium containing 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% non-ionic detergent Tween 20. polypeptide is carried out at 30°C. As a positive control for binding affinity, an anti-MHC class I monoclonal antibody can be used. . 123:342) with K _D 7 nM. A standard curve can be generated using serial dilutions of an anti-MHC class I monoclonal antibody. An anti-MHC class I co-immunomodulatory polypeptide or mAb is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces:

i) from the immobilized polypeptide (target); and ii) an internal reference layer.

Changing the number of molecules (analyte; eg, co-immunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a change in the interference pattern; this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of the interaction between the target and the analyte are the association constant (k _a ) and the dissociation constant (k _d ). The ratio of these two values (kd/ _a ) results in the affinity constant K _D . Determination of the binding affinity of a wild type immunomodulatory polypeptide (e.g., IL-2) to its receptor (e.g., IL-2R), as well as a variant immunomodulatory polypeptide (e.g., an IL-2 variant as described herein) to its recognized co-immunomodulatory polypeptide (eg, its receptor) (eg, IL-2R), thus allows you to determine the relative binding affinity of a variant co-immunomodulatory polypeptide compared to a wild-type co-immunomodulatory polypeptide for a recognized co-immunomodulatory polypeptide. That is, it can be determined whether the binding affinity of a variant immunomodulatory polypeptide to its receptor (its recognized immunomodulatory polypeptide) is reduced compared to the binding affinity of a wild-type immunomodulatory polypeptide to the same recognized immunomodulatory polypeptide, and if so, what is the percentage reduction in the binding affinity of the co-immunomodulatory polypeptide wild type.

The BLI assay is performed on a multiwell plate. For analysis, the plate loading scheme is determined, the analysis stages are determined, and biosensors are assigned in the Octet Data Acquisition software. The biosensor kit is hydrated. The hydrated biosensor kit and assay plate are equilibrated for 10 minutes on an Octet instrument. Once the data is received, the acquired data is loaded into the Octet data analysis software. The data is processed in the Processing window by specifying the standard subtraction method, y-alignment, inter-step correction, and Savitzky-Golay filtering. The data is analyzed in the analysis window by specifying the analysis steps (association and dissociation), the selection of the fitting model (1:1), the fitting method (global), and the window of interest (in seconds). The quality of the fit is evaluated. The K _D values for each data curve (analyte concentration) can be averaged if they are in the 3-fold range. The error values of K _D must be within the same order of magnitude of the affinity constant; R ² values must be greater than 0.95. See, for example, Abdiche et al. (2008) J. Anal. Biochem. 377:209.

In some instances, the ratio of: i) the binding affinity of a control modulating T cell multimeric polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to a recognized co-immunomodulatory polypeptide (e.g., IL-2R) to cells of a multimeric polypeptide of the present invention containing a wild-type immunomodulatory polypeptide variant (e.g., IL-2 variant) with a recognized immunomodulatory polypeptide (e.g., IL-2R) as measured by BLI (as described above) is at least 1 .5:1 at least

- 25 042111

2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100 :1 at least 500:1 at least

10 ² :1 at least 5x102:1 at least 10 ³ :1 at least 5x10 ³ :1 at least

10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In some instances, the ratio of: i) the binding affinity of a control T-cell modulating multimeric polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide) to a recognized communomodulatory polypeptide to ii) the binding affinity of a T-cell modulating multimeric polypeptide of the present invention containing a variant wild-type immunomodulatory polypeptide with a recognized co-immunomodulatory polypeptide, as measured by BLI is in the range from 1.5:1 to 10 ⁶ :1, for example, from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 10 ² :1, 10 ² :1 to 10 ³ :1, 10 ³ :1 to 10 ^{4 :1, 10 4 :} 1 to 10 ⁵ :1 or 10 ⁵ :1 to 10 ⁶ :1.

In some instances, the ratio of: i) the binding affinity of a control immunomodulatory polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to a cognate co-immunomodulatory polypeptide (e.g., IL-2R) to ii) the binding affinity of an immunomodulatory polypeptide of the present invention containing a wild-type immunomodulatory polypeptide variant (for example, an IL-2 variant) with a related immunomodulatory polypeptide (for example, IL-2R), as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, by at least 100:1, at least 500:1, at least ¹⁰² :1, at least 5x102:1, at least ¹⁰³ :1, at least ^5x103 :1, at least ¹⁰⁴ : 1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In some instances, the ratio of: i) the binding affinity of a reference immunomodulatory polypeptide (wherein the control comprises a wild-type immunomodulatory polypeptide) to a recognized co-immunomodulatory polypeptide to ii) the binding affinity of an immunomodulatory polypeptide of the present disclosure, comprising a variant wild-type immunomodulatory polypeptide to a recognized co-immunomodulatory polypeptide, as measured with using YOU, is in the range from 1.5:1 to 10 ⁶ :1, for example, from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 10 ² :1, from 10 ² :1 to 10 ³ :1, 10 ³ :1 to 10 ⁴ :1, 10 ⁴ :1 to 10 ⁵ :1 or 10 ⁵ :1 to 10 ⁶ :1.

IL-2/synTac

In some instances, the multimeric polypeptide contains wild-type (naturally occurring) IL-2 as a modulating domain. In some cases, the multimeric polypeptide contains a variant IL-2 polypeptide as a modulating domain.

A T-cell modulating multimeric polypeptide that contains an IL-2 polypeptide as a modulation domain (MOD) is also referred to as an IL-2/synTac, an IL-2/synTac polypeptide, or an IL2/multimeric polypeptide.

In some cases, the IL-2/synTac polypeptide contains a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide contains one copy of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide contains two copies of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide contains three copies of the wild-type IL-2 polypeptide. In some cases, the wild-type IL-2 polypeptide contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence depicted in FIG. 2A. The amino acid sequence of the wild-type human IL2 polypeptide can be as follows:

APTSSSTKKT

QLQLEHLLLD LQMILNGINN YKNPKLTRML TFI<FYMPI<I<A

TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1).

In some cases, the synTac polypeptide contains a variant IL-2 polypeptide. The IL-2 polypeptide variant present in the multimeric polypeptide exhibits reduced binding affinity for IL2R compared to the binding affinity of wild-type IL-2 for IL2R. A multimeric polypeptide that contains a variant IL-2 polypeptide also exhibits reduced binding affinity for IL2R compared to a control multimeric polypeptide containing wild-type IL-2 for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in Fig. 3A-3C).

In some instances, the IL-2/synTac polypeptide exhibits reduced binding affinity for IL2R compared to the binding affinity of an IL2 polypeptide containing the amino acid sequence depicted in FIG. 2A for IL2R. For example, in some instances, an IL2/synTac polypeptide binds IL2R with a binding affinity that is less than that of a control synTac polypeptide containing an IL2 polypeptide containing the amino acid sequence depicted in FIG. 2A for IL2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) shown in FIG. 3A-3C. For example, in some instances, an IL-2/synTac polypeptide binds IL2R with a binding affinity of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, at least 45%, and at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less binding affinity of a control synTac polypeptide containing an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A for IL2R (eg, IL2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C).

In some cases, the IL-2/synTac polypeptide has a binding affinity for IL2R that ranges from 100 nM to about 100 μM. In some instances, the IL-2/synTac polypeptide has an IL2R binding affinity that is between about 100 nM and 500 nM. For example, in some instances, an IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM from about 350 to about 400 nM, about 400 to about 450 nM, or about 450 to about 500 nM. In some instances, the IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that ranges from about 500 nM to 1 µM. For example, in some instances, an IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that is from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 to about 800 nM, from about 800 to about 900 nM, or from about 900 nM to about 1 μM. In some instances, the IL2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that ranges from about 1 μM to 10 µM. For example, in some instances, an IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that ranges from about 1 µM to 2 µM, about 2 µM to about 3 µM, about 3 µM to about 4 µM, about 4 µM to about 5 µM, about 5 µM to about 6 µM, about 6 µM to about 7 μM, from about 7 μM to about 8 μM, from about 8 μM to about 9 μM, or from about 9 μM to about 10 μM. In some instances, the IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that ranges from about 10 µM to 100 µM. For example, in some instances, an IL-2/synTac polypeptide has a binding affinity for IL2R (e.g., IL2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) that is from about 10 µM to about 20 µM, from about 20 µM to about 30 µM, from about 30 µM to about 40 µM, from about 40 µM to about 50 µM, from about 50 µM to about 60 µM, from about 60 µM up to about 70 μM, from about 70 μM to about 80 μM, from about 80 μM to about 90 μM, or from about 90 μM to about 100 μM.

An IL2 polypeptide variant present in an IL-2/synTac polypeptide may have one amino acid substitution relative to a wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as set forth in SEQ ID NO: 1). In some instances, the IL2 polypeptide variant present in an IL-2/synTac polypeptide has 2 to 10 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence shown in FIG. 2A or as shown in SEQ ID NO : 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 2 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 3 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the IL2 polypeptide variant present in the synTac polypeptide of the present invention has 4 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing

- 27 042111 containing the amino acid sequence shown in FIG. 2A or as shown in SEQ ID NO: 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 5 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 6 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 7 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as set forth in SEQ ID NO: 1). In some instances, the variant IL2 polypeptide present in the synTac polypeptide of the present invention has 8 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the IL2 polypeptide variant present in the synTac polypeptide of the present invention has 9 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1). In some instances, the IL2 polypeptide variant present in the synTac polypeptide of the present invention has 10 amino acid substitutions relative to the wild-type IL2 polypeptide (e.g., an IL2 polypeptide containing the amino acid sequence depicted in Figure 2A or as shown in SEQ ID NO: 1).

In some cases, the multimeric polypeptide of the present invention contains a first polypeptide and a second polypeptide, where the first polypeptide contains, in the direction from the amino-terminus (N-terminus) to the carboxyl-terminus (C-terminus):

a) epitope (eg, T-cell epitope);

b) the first major histocompatibility complex (MCHC) polypeptide, and

c) an immunomodulatory polypeptide (eg, an IL2 polypeptide variant of the present invention); and where the second polypeptide contains, in the direction from the N-terminus to the C-terminus:

a) a second MHC polypeptide; And

b) an immunoglobulin (Ig) Fc polypeptide.

In other cases, the multimeric polypeptide of the present invention contains a first polypeptide and a second polypeptide, where the first polypeptide includes, from the N-terminus to the C-terminus:

a) an epitope (eg, a T cell epitope); And

b) the first MHC polypeptide; and where the second polypeptide contains, in order from N-terminus to C-terminus:

a) an immunomodulatory polypeptide (eg, an IL2 polypeptide variant of the present invention);

b) a second MHC polypeptide; And

c) Fc Ig polypeptide.

In some instances, the first and second MHC polypeptides are class I MHC polypeptides; for example, in some instances, the first MHC polypeptide is a β2 microglobulin (B2M or B2M) MHC class I polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H-chain); or the first MHC polypeptide is the H chain of MHC class I and the second MHC polypeptide is the v2M MHC class I polypeptide). In other cases, the first and second MHC polypeptides are class II MHC polypeptides; for example, in some instances, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other instances, the first polypeptide is an MHC class II β-chain polypeptide and the second MHC polypeptide is an MHC class II α-chain polypeptide. In some instances, the multimeric polypeptide comprises two or more immunomodulatory polypeptides, wherein at least one of the immunomodulatory polypeptides is a variant of an IL2 immunomodulatory polypeptide of the present invention. When the multimeric polypeptide of the present invention includes two or more immunomodulatory polypeptides, in some cases, two or more immunomodulatory polypeptides are present in the same polypeptide chain and may be in tandem. When the multimeric polypeptide of the present invention includes two or more immunomodulatory polypeptides, in some cases, two or more immunomodulatory polypeptides are present in separate polypeptides. In some instances, the multimeric polypeptide of the present disclosure is a heterodimer. In some instances, the multimeric polypeptide of the present disclosure is

- 28 042111 is a trimeric polypeptide.

In some cases, the multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory domain (eg, an IL2 polypeptide variant of the present invention).

In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide; and ii) an immunomodulatory domain (eg, an IL2 polypeptide variant of the present invention).

In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an immunomodulatory domain (eg, an IL2 polypeptide variant of the present invention); and ii) a second MHC polypeptide.

In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain (eg, an IL2 polypeptide variant of the present invention); And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a second MHC polypeptide.

In some instances where the multimeric polypeptide of the present invention contains a non-Ig scaffold, the non-Ig scaffold is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

In some instances, the multimeric polypeptide of the present disclosure is monovalent. In some instances, the multimeric polypeptide of the present disclosure is multivalent. In some instances, the multivalent multimeric polypeptide of the present invention comprises an immunoglobulin Fc polypeptide on one of the first or second polypeptide. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present invention, the multimeric polypeptide may be a homodimer, where two multimeric polypeptide molecules are present in a homodimer, where two multimeric polypeptide molecules can be linked to each other by a disulfide bond, for example, through a polypeptide Fc present in two molecules. As another example, a multimeric polypeptide of the present invention may comprise three, four, or five multimeric polypeptide molecules, where the multimeric polypeptide molecules may be disulfide-linked to each other, such as through an Fc polypeptide present in the molecules.

In some cases, the multimeric polypeptide contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a b2M polypeptide; and iii) an IL2 polypeptide variant of the present invention; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) MHC class I heavy chain; and ii) an Fc polypeptide.

In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; And

- 29 042111 ii) β2M polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an IL2 polypeptide variant of the present invention;

ii) MHC class I heavy chain; and iii) an Fc polypeptide.

In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope;

ii) a b2M polypeptide;

iii) the first variant of the IL2 polypeptide of the present invention;

iv) a second variant of the IL2 polypeptide of the present invention; And

v) a third variant of the IL2 polypeptide of the present invention; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) MHC class I heavy chain; and ii) an Fc polypeptide.

In some instances, the first, second, and third variant IL2 polypeptides have the same amino acid sequence. In some instances, the first, second, and third variant IL2 polypeptides differ from each other in amino acid sequence. In some cases, the multimeric polypeptide of the present invention contains:

a) the first polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an epitope; and ii) a b2M polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) a first variant of the IL2 polypeptide of the present invention;

ii) a second variant of the IL2 polypeptide of the present invention; and iii) a third variant of the IL2 polypeptide of the present invention;

iv) MHC class I heavy chain; And

v) an Fc polypeptide.

In some instances, the first, second, and third variant IL2 polypeptides have the same amino acid sequence. In some instances, the first, second, and third variant IL2 polypeptides differ from each other in amino acid sequence.

F42 replacement.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence depicted. in fig. 2B, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2B, where amino acid 42 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2B, where amino acid 42 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2B, where amino acid 42 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2B, where amino acid 42 is Val. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2B, where amino acid 42 is Leu. In some cases, an IL-2 polypeptide variant present in

- 30 042111 multimeric polypeptide of the present disclosure, contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with respect to the amino acid sequence depicted in FIG. 2B, where amino acid 42 is Ile. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Y45 replacement.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is an amino acid other than tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is Val. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2F, where amino acid 45 is Leu. In some instances, an IL2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in FIG. 2F, where amino acid 45 is Ile. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric poly

- 31 042111 peptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Q126 replacement.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is an amino acid other than glutamine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Val. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Leu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2G, where amino acid 126 is Ile. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of an IL-2 polypeptide variant, such as when the three copies are in tandem with no linker between the three copies.

- 32 042111 or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42 and H16.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and where amino acid 16 is an amino acid other than histidine, for example, where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Ala and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Ala and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Val and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Leu and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2H, where amino acid 42 is Ile and amino acid 16 is Ala. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of an IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies or in a tandem.

- 33 042111 deme and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some cases, the multimeric polypeptide of the present invention contains 2 copies of an IL-2 variant containing F42A and H16A substitutions, where the multimeric polypeptide includes HLA class I and B2M heavy chain polypeptides, and where 2 copies of IL-2 (F42A, H16A) are located on the polypeptide chain containing an HLA class I heavy chain. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long. In some instances, a variant IL-2 polypeptide contains the amino acid sequence shown in FIG. 34V (containing H16A and F42A replacements).

Replacements for F42 and D20.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; and where amino acid 20 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; and where amino acid 20 is Asn, Gln, Lys, Arg, or His. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Val and amino acid 20 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Leu and amino acid 20 is Ala. In some cases, an IL-2 polypeptide variant,

- 34 042111 present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with respect to the amino acid sequence depicted in FIG. 2I, where amino acid 42 is Ile and amino acid 20 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Asn. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Gln. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Lys. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is Arg. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala and amino acid 20 is His. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20 and E15.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and where amino acid 15 is an amino acid

- 35 042111 other than glutamic acid, for example, where amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg , His or Asp. In some instances, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2J, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2I, where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2J, where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 is Ala. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid

- 36 042111 a sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in FIG. 2I, where amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some cases, when the IL2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL-2/ The synTac of the present invention contains a heavy chain of HLA class I and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20 and H16.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and where amino acid 16 is an amino acid other than histidine, for example, where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide at

- 37 042111 of the present disclosure, contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in FIG. 2K, where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2K, where amino acid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to

- 38 042111 about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20 and Q126.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is an amino acid other than phenylalanine, for example, where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and where amino acid 126 is an amino acid other than glutamine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg , His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; and where amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; and where amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2L, where amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20 and Y45.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and where amino acid 45 is an amino acid other than tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg , His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; and where amino acid 45 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; where is amino

- 40 042111 acid 45 is Ala, Gly, Val, Leu or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 45 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 45 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2M, where amino acid 42 is Ala, amino acid 20 is His, and amino acid 45 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- 2/synTac by

- 41 042111 of the present invention contains a heavy chain of HLA class I and β2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements F4, D20, Y45 and H16.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is an amino acid other than phenylalanine, for example, where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; where amino acid 45 is an amino acid other than tyrosine, for example, where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and where amino acid 16 is an amino acid other than histidine, for example, where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; where amino acid 45 is Ala, Gly, Val, Leu or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; where amino acid 45 is Ala, Gly, Val, Leu or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown

- 42 042111 in Fig. 2N, where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Val. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2N, where amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 16 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of an IL-2 polypeptide variant, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20, Y45 and Q126.

In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having the identity of ami

- 43 042111 no acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in FIG. 2O, where amino acid 42 is an amino acid other than phenylalanine, for example, where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; where amino acid 45 is an amino acid other than tyrosine, for example, where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and where amino acid 126 is an amino acid other than glutamine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg , His, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; where amino acid 45 is Ala, Gly, Val, Leu or Ile; and where amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; where amino acid 45 is Ala, Gly, Val, Leu or Ile; and where amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Val. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown

- 44 042111 in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 126 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2O, where amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 126 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM. from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM. In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements for F42, D20, Y45, H16 and Q126.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 20 is an amino acid other than aspartic acid, for example, where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; where amino acid 45 is an amino acid other than tyrosine, for example, where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 126 is an amino acid other than glutamine, for example, where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and where amino acid 16 is an amino acid other than histidine, for example, where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2p,

- 45 042111 where amino acid 42 is Ala, Gly, Val, Leu or Ile; where amino acid 20 is Ala, Gly, Val, Leu or Ile; where amino acid 45 is Ala, Gly, Val, Leu or Ile; where amino acid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg or His; where amino acid 45 is Ala, Gly, Val, Leu or Ile; where amino acid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%,

- 46 042111 at least 98% or at least 99% relative to the amino acid sequence shown in FIG. 2P, where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2P, where amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant containing it has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM . In some instances, the IL-2 polypeptide variant present in the multimeric polypeptide of the present disclosure is 133 amino acids long.

Replacements F42, Q126 and H16.

In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is an amino acid other than phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; where amino acid 126 is an amino acid other than glutamine, for example, where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and where amino acid 16 is an amino acid other than histidine, for example, where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 126 is Asn, Gln, Lys, Arg or His; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, vari

- 47 042111 ant of the IL-2 polypeptide present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with respect to the amino acid the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Val, amino acid 126 is Ala, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ile, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is Arg, and amino acid 16 is Ala. In some instances, an IL-2 polypeptide variant present in a multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 2Q, where amino acid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In some instances, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, where the two copies are in tandem without a linker between the two copies, or in tandem and separated by a linker peptide. In some instances, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, such as when the three copies are in tandem without a linker between the three copies, or in tandem and separated by a linker peptide. In some cases where the IL-2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL- The 2/synTac of the present invention contains an HLA class I heavy chain and b2M, the IL-2 polypeptide(s) is/are on the polypeptide chain containing the b2M polypeptide. In some instances, an IL-2 or synTac polypeptide variant comprising an IL-2 polypeptide variant has an IL2R binding affinity that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to at

- 48 042111 measured 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, about 25 µM to about 50 µM, about 50 µM to about 75 µM, or about 75 µM to about 100 µM . In some cases, the variant IL-2 polypeptide is 133 amino acids long.

4-1BBL.

In some cases, synTac suitable for use in the method of the present invention contains the 4-1BBL polypeptide as immunomodulatory domain(s). Suitable 4-1BBL immunomodulatory domains include the wild type 4-1BBL immunomodulatory domain and the variant 4-1BBL immunomodulatory domain.

The human wild type 4-1BBL amino acid sequence is shown in FIG. 36A. The homologous domain (THD) of human tumor necrosis factor (TNF) 4-1BBL includes amino acids 81-254, amino acids 80-254, or amino acids 80-246 of the amino acid sequence depicted in FIG. 36A. Thus, the amino acid sequence of wild-type human THD 4-1BBL can be, for example, one of SEQ ID NOs: 213-215, as follows:

PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT

KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA

LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:213).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:214).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO:215).

Wild-type 4-1BBL binds to 4-1BB (CD137). The amino acid sequence of 4-1BB is shown in FIG. 37. The 4-1BBL polypeptide variant of the present invention binds to 4-1BB with reduced affinity compared to wild-type 4-1BBL binding to 4-1BB.

Variant 4-1BBL polypeptides include polypeptides having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the corresponding wild-type 4-1BBL polypeptide and includes variants of 4-1BBL polypeptides that differ by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids or more 15 amino acids from the corresponding wild-type 4-1BBL polypeptide. In some instances, the 41BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by only one amino acid. In some instances, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 2 amino acids. In some instances, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 3 amino acids. In some instances, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 4 amino acids. In some instances, the 4-1BBL polypeptide variant differs in amino acid sequence from the wild-type 4-1BBL polypeptide by no more than 5 amino acids.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure exhibits reduced binding affinity for 4-1BB compared to the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A for 4-1BB. For example, in some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity less than the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A with the 4-1BB polypeptide containing the amino acid sequence depicted in FIG. 37. For example, in some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is at least 10% less, at least 15% less, at least 20% less. less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less,

- 49 042111 at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less binding affinity of a 41BBL polypeptide containing the amino acid sequence depicted in FIG. . 36A for 4-1BB (eg, the 4-1BB polypeptide containing the amino acid sequence depicted in FIG. 37).

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure exhibits reduced binding affinity for 4-1BB compared to the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence depicted in SEQ ID NO: 213 for 4-1BB . For example, in some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity lower than the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence depicted in SEQ ID NO: 213 for polypeptide 4 -1BB containing the amino acid sequence depicted in one of FIG. 37A-37C. For example, in some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure binds 4-1BB with a binding affinity that is at least 10% less, at least 15% less, at least 20% less. less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a 4-1BBL polypeptide containing the amino acid sequence depicted in SEQ ID NO: 213 for 4- 1BB (eg, a 4-1BB polypeptide containing the amino acid sequence depicted in Fig. 37).

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB that is between 100 nM and 100 μM. As another example, in some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide containing the amino acid sequence depicted in FIG. 37) that is about 100 nM to about 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 µM, about 1 µM to about 5 µM, about 5 µM to about 10 µM, about 10 µM to about 15 µM, about 15 µM to about 20 µM, about 20 µM to about 25 µM, from about 25 µM to about 50 µM, from at approximately 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure exhibits increased production in a mammalian host cell compared to production in the same mammalian host cell of a control multimeric polypeptide containing a 4-1BBL polypeptide (e.g., polypeptide 4 -1BBL containing the amino acid sequence depicted in Fig. 36A or as shown in SEQ ID NO: 213). For example, in some instances, a 4-1BBL variant polypeptide present in a multimeric polypeptide, when expressed in a mammalian host cell, is produced in an amount that is from 25% higher to about 50% higher, from about 50% higher to about 75% higher. % higher, from about 75% higher to about 2 times higher, from about 2 times higher to about 5 times higher, from about 5 times higher to about 10 times higher, from about 10 times higher to about 20 times higher, about 20 times higher to about 30 times higher, from about 30 times higher to about 40 times higher, from about 40 times higher to about 50 times higher, from about 50 times higher up to about 75 times higher, from about 75 times higher to about 100 times higher, or more than 100 times higher than the amount of a control multimeric polypeptide containing a wild-type 4-1BBL polypeptide (for example, a 4-1BBL polypeptide containing the amino acid sequence, shown in Fig. 36A or as shown in SEQ I D NO: 213) obtained in the same mammalian host cell.

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide is produced in a mammalian host cell in an amount of from about 50 mg/L to about 75 mg/L, from about 75 mg/L to about 100 mg/L, from about 100 mg/L to about 150 mg/L, about 150 mg/L to about 200 mg/L, about 200 mg/L to about 250 mg/L, about 250 mg/L to about 500 mg/L, or more than 500 mg/l. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide is produced in a mammalian host cell in an amount

- 50 042111 from about 10 mg/l to about 15 mg/l, from about 15 mg/l to about 20 mg/l, from about 20 mg/l to about 25 mg/l, from about 25 mg/l to about 30 mg/l, from about 35 mg/l to about 40 mg/l, from about 40 mg/l to about 45 mg/l, or from about 45 mg/l to about 50 mg/l.

A 4-1BBL polypeptide variant present in a multimeric polypeptide may have one amino acid substitution relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A or as shown in SEQ ID NO: 213) . In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 2 to 10 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide containing the amino acid sequence depicted in Figure 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 2 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 41BBL polypeptide containing the amino acid sequence depicted in FIG. 36A or as shown in SEQ ID NO : 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 3 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 4 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence depicted in Figure 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 5 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 6 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence depicted in Figure 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant of the present disclosure has 7 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A or as shown in SEQ ID NO: 213) . In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 8 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 9 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence depicted in FIG. 36A or as shown in SEQ ID NO: 213). In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 10 amino acid substitutions relative to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A or as shown in SEQ ID NO: 213).

In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure has 11 to 50 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide containing the amino acid sequence depicted in Figure 36A or as shown in SEQ ID NO: 213). For example, in some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure has 11 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45 or 45 to 50 amino acid substitutions relative to a wild-type 4-1BBL polypeptide (eg, a 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A or as shown in SEQ ID NO: 213).

Suitable variants of the 4-1BBL polypeptides that can be included in the multimeric polypeptide of the present invention include those described above.

4-1BBL with K127 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Tip, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu. In some instances, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90% amino acid sequence identity,

- 51 042111 at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly , Ala, Val, Leu, Ile,

Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp, or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid change in K48. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at K48. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid change in K48. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in K48.

Replacements K127 + M91.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at M91 wherein amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 91 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 11 is different from methionine, for example, where amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 11 is Ala.

Replacements K127 + F92.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution in F92, where amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 92 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; And

- 52 042111 ii) amino acid 12 is different from phenylalanine, for example, where amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 12 is Ala.

Replacements K127 + Q94.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at Q94, where amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 94 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 14 is different from glutamine, for example, where amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 14 is Ala.

Replacements K127 + L95.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at L95, where amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 95 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 15 is different from leucine, for example, where amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 15 is Ala.

Replacements K127 + V96.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution in V96, where amino acid 96 is Gly, Ala, Leu, Ile, Pro,

- 53 042111

Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 127 is Ala; and amino acid 96 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 16 is other than valine, for example, where amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 16 is Ala.

Replacements K127 + Q98.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at Q98, where amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 98 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 18 is different from glutamine, for example, where amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 18 is Ala.

Replacements K127+N99.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at N99, where amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 99 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 19 is different from asparagine, for example, where amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 19 is Ala.

- 54 042111

Replacements K127 + V100.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at V100, where amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 100 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 20 is other than valine, for example, where amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 20 is Ala.

Replacements K127 + L101.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at L101 wherein amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 101 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 21 is different from leucine, for example, where amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 21 is Ala.

Replacements for K127+ L102.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at LI02 wherein amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 102 is Ala. In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 22 is different from leucine, for example, where amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 22 is Ala.

Replacement for K127+1103.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at I103, where amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 103 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 23 is different from isoleucine, for example, where amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 23 is Ala.

Replacements K127 + D104.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at D104 wherein amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 104 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg , His, Asp or Glu; and ii) amino acid 24 is different from aspartic acid, for example, where amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg , His or Glu.

In some cases, amino acid 47 is Ala; and amino acid 24 is Ala.

Replacements K127 + G105.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 56 042111

98% or at least 99% relative to the amino acid sequence depicted in FIG. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys,

Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at G105, where amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 105 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 25 is different from glycine, for example, where amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 25 is Ala.

Replacements K127 + P106.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at P106 wherein amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 106 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 26 is different from proline, for example, where amino acid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 26 is Ala.

Replacements K127 + L107.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at L107 wherein amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 107 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino-

- 57 042111 acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg,

His, Asp or Glu; and ii) amino acid 27 is different from leucine, for example, where amino acid 27 is Gly,

Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, amino acid 47 is Ala; and amino acid 27 is Ala.

Replacements K127 + S108.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid change to S108, where amino acid 108 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 108 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 28 is different from serine, for example, where amino acid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 28 is Ala.

Replacements K127 + W109.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution in W109, where amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 109 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 29 is different from tryptophan, for example, where amino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 29 is Ala.

Replacements K127 + Y110.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys,

- 58 042111

Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at Y110, where amino acid 110 is Gly, Ala, Val, Leu, Ile,

Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu.

In some cases, amino acid 127 is Ala; and amino acid 110 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 30 is different from tyrosine, for example, where amino acid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala; and amino acid 30 is Ala.

Replacements K127 + S111.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid change to S111, where amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 111 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 31 is different from serine, for example, where amino acid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 31 is Ala.

Replacements K127 + D112.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at D112 wherein amino acid 112 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 112 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg , His, Asp or Glu; and ii) amino acid 32 is different from aspartic acid, for example, where amino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg , His or Glu.

In some cases, amino acid 47 is Ala; and amino acid 32 is Ala.

Replacements K127 + P113.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at P113, where amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 113 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 33 is different from proline, for example, where amino acid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 33 is Ala.

Replacements K127 + G114.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at G114, where amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 114 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 34 is different from glycine, for example, where amino acid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 34 is Ala.

Replacements K127 + L115.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where:

i) amino acid 127 (denoted by x) is an amino acid other than lysine, for example, where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) an amino acid substitution at L115, where amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu .

In some cases, amino acid 127 is Ala; and amino acid 115 is Ala. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where:

i) amino acid 47 is an amino acid other than lysine, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; and ii) amino acid 35 is different from leucine, for example, where amino acid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, amino acid 47 is Ala; and amino acid 35 is Ala.

4-1BBL with Q227 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 147 is different from glutamine, for example, where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in Q148. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Q148. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in Q148. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at Q148.

4-1BBL with M91 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36E, where amino acid 91 (denoted x) is an amino acid other than methionine, e.g., where amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 11 is other than methionine, for example, where amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Asn, Gln , Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in M12. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at M12. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having amino acid identity

- 61 042111 sequences of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215, with an amino acid substitution in M12. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at M12.

4-1BBL with F92 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36F, where amino acid 92 (denoted x) is an amino acid other than phenylalanine, e.g., where amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 12 is other than phenylalanine, for example, where amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in F13. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in F13. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in F13. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in F13.

4-1BBL with Q94 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36G, where amino acid 94 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 14 is other than glutamine, for example, where amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at Q15. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at Q15. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at Q15. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at Q15.

4-1BBL with L95 replacement.

- 62 042111

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36H, where amino acid 95 (denoted by x) is an amino acid other than leucine, for example, where amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 15 is other than leucine, for example, where amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at L16. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L16. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at L16. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L16.

4-1BBL with V96 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36I, where amino acid 96 (denoted x) is an amino acid other than valine, e.g., where amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 16 is other than valine, for example, where amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V17. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at V17. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V17. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at V17.

4-1BBL with Q98 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36J, where amino acid 98 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,

- 63 042111

Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 18 is other than glutamine, for example, where amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in Q19. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at Q19. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in Q19. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at Q19.

4-1BBL with N99 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36K, where amino acid 99 (denoted by x) is an amino acid other than asparagine, e.g., where amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 19 is different from asparagine, for example, where amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Gln , Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in N20. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at N20. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in N20. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at N20.

4-1BBL with V100 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36L, where amino acid 100 (denoted x) is an amino acid other than valine, e.g., where amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 20 is other than valine, for example, where amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, glen,

- 64 042111

Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V21. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V21. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V21. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at V21.

4-1BBL with L101 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36M, where amino acid 101 (denoted by x) is an amino acid other than leucine, e.g., where amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 21 is other than leucine, for example, where amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L22. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L22. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at L22. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L22.

4-1BBL with L102 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36N, where amino acid 102 (denoted by x) is an amino acid other than leucine, for example, where amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 22 is other than leucine, for example, where amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid change in L23. In some cases, the 4-1BBL polypeptide variant,

- 65 042111 present in the multimeric polypeptide of the present disclosure, contains the amino acid sequence shown in SEQ ID NO: 214, with an amino acid substitution in L23. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid change in L23. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L23.

4-1BBL with I103 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 360, where amino acid 103 (denoted x) is an amino acid other than isoleucine, e.g., where amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 23 is other than isoleucine, for example, where amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with amino acid substitution in I24. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at I24. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with amino acid substitution in I24. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at I24.

4-1BBL with D104 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36P, where amino acid 104 (denoted x) is an amino acid other than aspartic acid, e.g., where amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 24 is different from aspartic acid, for example, where amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in D25. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at D25. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215, with

- 66 042111 amino acid substitution in D25. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at D25.

4-1BBL with G105 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36Q, where amino acid 105 (denoted x) is an amino acid other than glycine, e.g., where amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 25 is other than glycine, e.g., where amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution at G26. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G26. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution at G26. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G26.

4-1BBL with P106 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36R, where amino acid 106 (denoted x) is an amino acid other than proline, e.g., where amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 26 is other than proline, for example, where amino acid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in P27. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at P27. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in P27. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in P27.

4-1BBL with L107 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having the identity

- 67 042111 amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in FIG. 36S, where amino acid 107 (denoted x) is an amino acid other than leucine, e.g., where amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 27 is other than leucine, for example, where amino acid 27 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L28. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L28. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L28. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L28.

4-1BBL with S108 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36T, where amino acid 108 (denoted x) is an amino acid other than serine, e.g., where amino acid 108 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 28 is other than serine, for example, where amino acid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S29. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at S29. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S29. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at S29.

4-1BBL with W109 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36U, where amino acid 109 (denoted by x) is an amino acid other than tryptophan, e.g., where amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 29 is other than tryptophan, e.g., where amino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys,

Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in W30. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in W30. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in W30. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in W30.

4-1BBL with Y110 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36V, where amino acid 110 (denoted x) is an amino acid other than tyrosine, e.g., where amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 30 is other than tyrosine, for example, where amino acid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution at Y31. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Y31. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution at Y31. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at Y31.

4-1BBL with S111 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36W, where amino acid 111 (denoted x) is an amino acid other than serine, e.g., where amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 31 is other than serine, for example, where amino acid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 69 042111 according to the present disclosure, contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 214, with an amino acid substitution at S32. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at S32. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S32. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at S32.

4-1BBL with D112 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36X, where amino acid 112 (denoted by x) is an amino acid other than aspartic acid, e.g., where amino acid 112 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 32 is different from aspartic acid, for example, where amino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in D33. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at D33. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in D33. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at D33.

4-1BBL with P113 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36Y, where amino acid 113 (denoted by x) is an amino acid other than proline, e.g., where amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 33 is other than proline, for example, where amino acid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in P34. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at P34. In some

- 70 042111 cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in P34. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at P34.

4-1BBL with G114 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36Z, where amino acid 114 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 34 is other than glycine, e.g., where amino acid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in G35. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G35. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in G35. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G35.

4-1BBL with L115 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36AA, where amino acid 115 (denoted x) is an amino acid other than leucine, e.g., where amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 35 is other than leucine, for example, where amino acid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L36. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L36. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L36. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence

- 71 042111 presented in SEQ ID NO: 215, with an amino acid substitution in L36.

4-1BBL with G117 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36BB, where amino acid 117 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 117 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 37 is other than glycine, for example, where amino acid 37 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in G38. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G38. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G38. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G38.

4-1BBL with V118 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36CC, where amino acid 118 (denoted by x) is an amino acid other than valine, e.g., where amino acid 118 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 38 is other than valine, for example, where amino acid 38 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V39. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V39. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V39. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in V39.

4-1BBL with S119 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in

- 72 042111 fig. 36DD, where amino acid 119 (denoted x) is an amino acid other than serine, e.g., where amino acid 119 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 39 is other than serine, for example, where amino acid 39 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S40. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at S40. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S40. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at S40.

4-1BBL with L120 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36EE, wherein amino acid 120 (denoted by x) is an amino acid other than leucine, e.g., where amino acid 120 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 40 is other than leucine, for example, where amino acid 40 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid change in L41. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L41. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid change in L41. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L41.

4-1BBL with T121 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36FF, where amino acid 121 (denoted x) is an amino acid other than threonine, e.g., where amino acid 121 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence.

- 73 042111 of the sequence shown in SEQ ID NO: 213, where amino acid 41 is other than threonine, for example, where amino acid 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met ,

Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at T42. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at T42. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at T42. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at T42.

4-1BBL with G122 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36GG, where amino acid 122 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 122 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 42 is other than glycine, for example, where amino acid 42 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr Cys, Asn, Gln, Lys , Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in G43. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G43. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G43. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G43.

4-1BBL with G123 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36HH, where amino acid 123 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 123 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 43 is other than glycine, for example, where amino acid 43 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr Cys, Asn, Gln, Lys , Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

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98% or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution in G44. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G44. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G44. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G44.

4-1BBL with L124 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36II, where amino acid 124 (denoted by x) is an amino acid other than leucine, for example, where amino acid 124 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 44 is other than leucine, for example, where amino acid 44 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid change in L45. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L45. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at L45. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L45.

4-1BBL with S125 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36JJ, where amino acid 125 (denoted x) is an amino acid other than serine, e.g., where amino acid 125 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 45 is other than serine, for example, where amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S46. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at S46. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having amino acid identity

- 75 042111 sequences of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215, with an amino acid substitution in S46. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at S46.

4-1BBL with Y126 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36KK, where amino acid 126 (denoted by x) is an amino acid other than tyrosine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 46 is other than tyrosine, for example, where amino acid 46 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution at Y47. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Y47. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution at Y47. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at Y47.

4-1BBL with E128 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36LL, where amino acid 128 (denoted x) is an amino acid other than glutamic acid, e.g., where amino acid 128 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 48 is different from glutamic acid, for example, where amino acid 48 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His or Asp.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in E49. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at E49. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in E49. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at E49.

4-1BBL with D129 replacement.

- 76 042111

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36MM, where amino acid 129 (denoted by x) is an amino acid other than aspartic acid, e.g., where amino acid 129 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 49 is different from aspartic acid, for example, where amino acid 49 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in D50. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at D50. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in D50. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at D50.

4-1BBL with T130 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36NN, where amino acid 130 (denoted x) is an amino acid other than threonine, e.g., where amino acid 130 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 50 is other than threonine, for example, where amino acid 50 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at T51. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at T51. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at T51. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at T51.

4-1BBL with K131 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36OO, where amino acid 131 (denoted x) is an amino acid other than lysine, e.g., where amino acid 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,

- 77 042111

Cys, Met, Asn, Gln, Arg, His, Asp, or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 51 is other than lysine, e.g., where amino acid 51 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid change in K52. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at K52. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid change in K52. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at K52.

4-1BBL with E132 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36PP where amino acid 132 (denoted x) is an amino acid other than glutamic acid, e.g. where amino acid 132 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 52 is different from glutamic acid, for example, where amino acid 52 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His or Asp.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in E53. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at E53. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in E53. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at E53.

4-1BBL with F144 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36QQ, where amino acid 144 (denoted by x) is an amino acid other than phenylalanine, e.g., where amino acid 144 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 64 is other than phenylalanine, for example, where amino acid 64 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,

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Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in F65. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in F65. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in F65. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in F65.

4-1BBL with F145 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36RR, where amino acid 145 (denoted x) is an amino acid other than phenylalanine, e.g., where amino acid 145 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 65 is other than phenylalanine, for example, where amino acid 65 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in F66. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in F66. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in F66. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in F66.

4-1BBL with Q146 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36SS, where amino acid 146 (denoted by x) is an amino acid other than glutamine, e.g., where amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 66 is other than glutamine, for example, where amino acid 66 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in Q67. In some cases, the 4-1BBL polypeptide variant,

- 79 042111 present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Q67. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in Q67. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at Q67.

4-1BBL with L147 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36TT, where amino acid 147 (denoted by x) is an amino acid other than leucine, for example, where amino acid 147 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 67 is other than leucine, for example, where amino acid 67 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L68. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L68. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L68. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L68.

4-1BBL with E148 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36UU where amino acid 148 (denoted x) is an amino acid other than glutamic acid, e.g. where amino acid 148 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 68 is different from glutamic acid, for example, where amino acid 68 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His or Asp.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in E69. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at E69. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215, with

- 80 042111 amino acid substitution in E69. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at E69.

4-1BBL with L149 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36VV, where amino acid 149 (denoted by x) is an amino acid other than leucine, for example, where amino acid 149 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 69 is other than leucine, for example, where amino acid 69 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L70. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L70. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid change in L70. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at L70.

4-1BBL with R150 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36WW, where amino acid 150 (denoted by x) is an amino acid other than arginine, e.g., where amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. sequence shown in SEQ ID NO: 213, where amino acid 70 is other than arginine, for example, where amino acid 70 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn , Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R71. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R71. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in R71. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R71.

4-1BBL with R151 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having the identity

- 81 042111 amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in FIG. 36XX, where amino acid 151 (denoted by x) is an amino acid other than arginine, e.g., where amino acid 151 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 71 is other than arginine, for example, where amino acid 71 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R72. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R72. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in R72. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R72.

4-1BBL with V152 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36YY, where amino acid 152 (denoted by x) is an amino acid other than valine, e.g., where amino acid 152 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 72 is other than valine, e.g., where amino acid 72 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V73. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V73. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V73. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in V73.

4-1BBL with V153 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36ZZ, where amino acid 153 (denoted x) is an amino acid other than valine, e.g., where amino acid 153 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in a multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 73 is other than valine, e.g., where amino acid 73 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln,

Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V74. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V74. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V74. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in V74.

4-1BBL with G155 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36AAA, where amino acid 155 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 155 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 75 is other than glycine, for example, where amino acid 75 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in G76. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G76. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G76. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G76.

4-1BBL with E156 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36BBB, where amino acid 156 (denoted by x) is an amino acid other than glutamic acid, e.g., where amino acid 156 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 41BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence, shown in SEQ ID NO: 213, where amino acid 76 is different from glutamic acid, for example, where amino acid 76 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His or Asp.

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 83 042111 according to the present disclosure, contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 214, with amino acid substitution in E77. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at E77. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in E77. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at E77.

4-1BBL with G157 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36CCC, where amino acid 157 (denoted x) is an amino acid other than glycine, e.g., where amino acid 157 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 77 is other than glycine, for example, where amino acid 77 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in G78. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G78. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G78. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G78.

4-1BBL with S158 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36DDD, where amino acid 158 (denoted by x) is an amino acid other than serine, e.g., where amino acid 158 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 78 is other than serine, for example, where amino acid 78 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S79. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at S79. In some

- 84 042111 cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution in S79. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at S79.

4-1BBL with D184 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36EEE, where amino acid 184 (denoted x) is an amino acid other than aspartic acid, e.g., where amino acid 184 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 104 is different from aspartic acid, for example, where amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in D105. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at D105. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in D105. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at D105.

4-1BBL with L185 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36FFF, where amino acid 185 (denoted x) is an amino acid other than leucine, e.g., where amino acid 185 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 105 is other than leucine, for example, where amino acid 105 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L106. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at L106. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L106. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence

- 85 042111 presented in SEQ ID NO: 215, with an amino acid substitution in L106.

4-1BBL with P186 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36GGG, where amino acid 186 (denoted x) is an amino acid other than proline, e.g., where amino acid 186 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 106 is other than proline, for example, where amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in P107. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in P107. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in P107. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in P107.

4-1BBL with P187 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36HHH, where amino acid 187 (denoted x) is an amino acid other than proline, e.g., where amino acid 187 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 107 is other than proline, for example, where amino acid 107 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in P108. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at P108. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in P108. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at P108.

4-1BBL with S189 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in

- 86 042111 fig. 36III, where amino acid 189 (denoted by x) is an amino acid other than serine, e.g., where amino acid 189 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the 4-1 BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 109 is other than serine, for example, where amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn , Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S110. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in S110. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S110. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in S110.

4-1BBL with S190 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36JJJ, where amino acid 190 (denoted by x) is an amino acid other than serine, e.g., where amino acid 190 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 110 is other than serine, for example, where amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S111. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in S111. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S111. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in S111.

4-1BBL with E191 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36KKK where amino acid 191 (denoted by x) is an amino acid other than glutamic acid, e.g. where amino acid 191 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% relative amino acid sequence identity. 87 042111 but the amino acid sequence shown in SEQ ID NO: 213, where amino acid 111 is different from glutamic acid, for example, where amino acid 111 is Gly, Ala, Val, Leu,

Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Asp.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in E112. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in E112. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in E112. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in E112.

4-1BBL with R193 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36LLL, where amino acid 193 (denoted by x) is an amino acid other than arginine, for example, where amino acid 193 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 113 is different from arginine, for example, where amino acid 113 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R114. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R114. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in R114. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R114.

4-1BBL with N194 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36MMM, where amino acid 194 (denoted by x) is an amino acid other than asparagine, e.g., where amino acid 194 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 114 is different from asparagine, for example, where amino acid 114 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Gln , Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least

- 88 042111

98% or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution in N115. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at N115. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in N115. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at N115.

4-1BBL with S195 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36NNN, where amino acid 195 (denoted by x) is an amino acid other than serine, e.g., where amino acid 195 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 115 is other than serine, for example, where amino acid 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in S116. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in S116. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in S116. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at S116.

4-1BBL with F197 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36OOO, where amino acid 197 (denoted x) is an amino acid other than phenylalanine, e.g., where amino acid 197 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 117 is other than phenylalanine, for example, where amino acid 117 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in F118. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in F118. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having amino acid identity

- 89 042111 sequences of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215, with the amino acid substitution in F118. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution in F118.

4-1BBL with Q210 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36PPP, where amino acid 210 (denoted by x) is an amino acid other than glutamine, for example, where amino acid 210 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 130 is other than glutamine, for example, where amino acid 130 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in Q131. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Q131. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in Q131. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at Q131.

4-1BBL with R211 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36QQQ, where amino acid 211 (denoted by x) is an amino acid other than arginine, for example, where amino acid 211 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 131 is other than arginine, for example, where amino acid 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn , Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R132. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R132. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in R132. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R132.

4-1BBL with L212 replacement.

- 90 042111

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36RRR, where amino acid 212 (denoted by x) is an amino acid other than leucine, for example, where amino acid 212 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 132 is other than leucine, for example, where amino acid 132 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L133. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at L133. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L133. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at L133.

4-1BBL with G213 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36SSS, where amino acid 213 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 213 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 133 is other than glycine, for example, where amino acid 133 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in G134. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G134. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G134. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G134.

4-1BBL with V214 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36TTT, where amino acid 214 (denoted by x) is an amino acid other than valine, e.g., where amino acid 214 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,

- 91 042111

Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 134 is other than valine, for example, where amino acid 134 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V135. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V135. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in V135. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in V135.

4-1BBL with replacement H215.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36UUU, where amino acid 215 (denoted by x) is an amino acid other than histidine, for example, where amino acid 215 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 135 is different from histidine, for example, where amino acid 135 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in H136. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at H136. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in H136. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at H136.

4-1BBL with L216 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36VVV, where amino acid 216 (denoted by x) is an amino acid other than leucine, for example, where amino acid 216 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 136 is other than leucine, for example, where amino acid 136 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,

- 92 042111

Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L137. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at L137. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L137. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at L137.

4-1BBL with H217 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36WWW, where amino acid 217 (denoted by x) is an amino acid other than histidine, e.g., where amino acid 217 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 137 is different from histidine, for example, where amino acid 137 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in H138. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at H138. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in H138. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at H138.

4-1BBL with T218 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36XXX, where amino acid 218 (denoted by x) is an amino acid other than threonine, for example, where amino acid 218 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 138 is other than threonine, for example, where amino acid 138 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in T139. In some cases, the 4-1BBL polypeptide variant,

- 93 042111 present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at T139. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in T139. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at T139.

4-1BBL with E219 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36YYY where amino acid 219 (denoted x) is an amino acid other than glutamic acid, e.g. where amino acid 219 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 139 is different from glutamic acid, for example, where amino acid 139 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met , Asn, Gln, Lys, Arg, His or Asp.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in E140. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at E140. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in E140. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at E140.

4-1BBL with R221 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36ZZZ, where amino acid 221 (denoted by x) is an amino acid other than arginine, for example, where amino acid 221 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 141 is other than arginine, for example, where amino acid 141 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn , Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R142. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R142. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. sequence shown in SEQ ID NO: 215, with

- 94 042111 amino acid substitution in R142. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R142.

4-1BBL with R223 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36AAAA where amino acid 223 (denoted by x) is an amino acid other than arginine, e.g. where amino acid 223 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 143 is other than arginine, for example, where amino acid 143 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser Thr, Cys, Met, Asn , Gln, Lys, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in R144. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in R144. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in R144. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution in R144.

4-1BBL with H224 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36BBBB, where amino acid 224 (denoted x) is an amino acid other than histidine, e.g., where amino acid 224 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 144 is different from histidine, for example, where amino acid 144 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in H145. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at H145. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in H145. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at H145.

4-1BBL with W226 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having the identity

- 95 042111 amino acid sequence of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence depicted in FIG. 36CCCC, where amino acid 226 (denoted x) is an amino acid other than tryptophan, e.g., where amino acid 226 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 146 is other than tryptophan, for example, where amino acid 146 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with the amino acid substitution in W147. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution in W147. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in W147. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution in W147.

4-1BBL with L228 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36DDDD, where amino acid 228 (denoted by x) is an amino acid other than leucine, for example, where amino acid 228 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 148 is other than leucine, for example, where amino acid 148 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in L149. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at L149. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in L149. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at L149.

4-1BBL with T229 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36EEEE, where amino acid 229 (denoted x) is an amino acid other than threonine, e.g., where amino acid 229 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid

- 96 042111 a sequence having an amino acid sequence identity of at least

90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 213, where amino acid 149 is other than threonine, e.g., where amino acid 149 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,

Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at T150. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution at T150. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution at T150. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with an amino acid substitution at T150.

4-1BBL with Q230 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36FFFF, where amino acid 230 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 230 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 150 is other than glutamine, for example, where amino acid 150 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in Q151. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at Q151. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with an amino acid substitution in Q151. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at Q151.

4-1BBL with G231 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36GGGG, where amino acid 231 (denoted by x) is an amino acid other than glycine, e.g., where amino acid 231 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 151 is other than glycine, for example, where amino acid 151 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide

- 97 042111 according to the present disclosure, contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 214, with amino acid substitution in G152. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at G152. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution in G152. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at G152.

4-1BBL with T233 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36HHHH, where amino acid 233 (denoted by x) is an amino acid other than threonine, e.g., where amino acid 233 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 153 is other than threonine, for example, where amino acid 153 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution at T154. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with the amino acid substitution at T154. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 215 with the amino acid substitution at T154. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution at T154.

4-1BBL with V234 replacement.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in Fig. 36IIII, where amino acid 234 (denoted x) is an amino acid other than valine, e.g., where amino acid 234 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 213, where amino acid 154 is other than valine, for example, where amino acid 154 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the amino acid sequence. the sequence shown in SEQ ID NO: 214 with an amino acid substitution in V155. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 214 with an amino acid substitution in V155. In some

- 98 042111 cases, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution in V155. In some instances, the 4-1BBL polypeptide variant present in the multimeric polypeptide of the present disclosure contains the amino acid sequence shown in SEQ ID NO: 215 with the amino acid substitution in V155.

Exemplary multimeric polypeptides containing the immunomodulatory polypeptide 4-1BBL.

Exemplary multimeric polypeptides that are suitable for use in the method of the present invention are described below.

K127.

In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a b2M polypeptide; and iii) a 4-1BBL polypeptide variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence depicted in FIG. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; or a 4-1BBL polypeptide variant of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence specified in SEQ ID NO : 213, with an amino acid change at K47, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Arg, His, Asp or Glu; and b) a second polypeptide comprising, in the N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide comprising in the N-terminal to C-terminal direction: i) a 4-1BBL polypeptide variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence depicted in FIG. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; or a 4-1BBL polypeptide variant of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence specified in SEQ ID NO : 213, with an amino acid change at K47, e.g., where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Arg, His, Asp or Glu; ii) MHC class I heavy chain; and iii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a b2M polypeptide; iii) a first variant of the 4-1BBL polypeptide of the present invention; iv) a second variant of the 4-1BBL polypeptide of the present invention; and v) a third variant of the 4-1BBL polypeptide of the present invention; and b) a second polypeptide comprising in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence, shown in FIG. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; for example, each of the first, second, and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence represented by in SEQ ID NO: 213, with an amino acid change in K47, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide containing in the direction from the N-terminus to the C-terminus: i) the first variant of the 4-1BBL polypeptide of the present invention; ii) a second variant of the 4-1BBL polypeptide of the present invention; and iii) a third variant of the 4-1BBL polypeptide of the present invention; iv) MHC class I heavy chain; and v) an Fc polypeptide. In some cases, each of the first, second and third variants of polypeptides 4

- 99 042111

1BBL contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; for example, each of the first, second, and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence represented by in SEQ ID NO: 213, with an amino acid change in K47, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu. In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an M91 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an F92 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a Q94 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L95 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a V96 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a Q98 substitution (based on the numbering shown in FIG. 36A). In some instances, the 41BBL polypeptide variant contains a K127 substitution and an N99 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a V100 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L101 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L102 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an I103 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a D104 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a G105 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a P106 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L107 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an S108 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a W109 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a Y110 substitution (based on the numbering shown in Figure 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an S111 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a D112 substitution (based on the numbering shown in Figure 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a PIZ substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a G114 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L115 substitution (based on the numbering shown in FIG. 36A).

In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide containing in the direction from the N-terminus to the C-terminus: i) the first variant of the 4-1BBL polypeptide of the present invention; ii) a linker; iii) a second variant of the 4-1BBL polypeptide of the present invention; iv) a linker; v) a third variant of the 4-1BBL polypeptide of the present invention; vi) MHC class I heavy chain; and vii) an Fc polypeptide. In some cases, each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence, shown in FIG. 36B, where amino acid 127 (denoted x) is an amino acid other than lysine, e.g., where amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu; for example, each of the first, second, and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence represented by in SEQ ID NO: 213, with an amino acid change in K47, for example, where amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu. In some cases, the linker contains the sequence (GSSSS) _n (SEQ ID NO: 131), where n is 1, 2, 3, 4, or 5. In some embodiments, n is 4. In some embodiments, n is 5. In some cases, n is the 4-1BBL polypeptide variant contains a K127 substitution and an M91 substitution (based on the numbering shown

- 100 042111 in Fig. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an F92 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a Q94 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L95 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a V96 substitution (based on the numbering shown in FIG. 36A). In some instances, the 41BBL polypeptide variant contains a K127 substitution and a Q98 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an N99 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a V100 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L101 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L102 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an I103 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a D104 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a G105 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a P106 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L107 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an S108 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a W109 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a Y110 substitution (based on the numbering shown in Figure 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an S111 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a D112 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a P113 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and a G114 substitution (based on the numbering shown in FIG. 36A). In some instances, the 4-1BBL polypeptide variant contains a K127 substitution and an L115 substitution (based on the numbering shown in FIG. 36A).

Q227.

In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a b2M polypeptide; and iii) a 4-1BBL polypeptide variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence depicted in FIG. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or a 4-1BBL polypeptide variant of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence specified in SEQ ID NO : 213, with amino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys Arg, His, Asp or Glu; and b) a second polypeptide comprising in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide comprising in the N-terminal to C-terminal direction: i) a 4-1BBL polypeptide variant comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity relative to the amino acid sequence depicted in FIG. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or a 4-1BBL polypeptide variant of the present invention comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence specified in SEQ ID NO : 213, with amino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys Arg, His, Asp or Glu; ii) MHC class I heavy chain; and iii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction

- 101 042111 N-terminus to C-terminus: i) epitope; ii) a β2M polypeptide; iii) a first variant of the 4-1BBL polypeptide of the present invention; iv) a second variant of the 4-1BBL polypeptide of the present invention; and v) a third variant of the 4-1BBL polypeptide of the present invention; and b) a second polypeptide comprising in N-terminal to C-terminal direction: i) an MHC class I heavy chain; and ii) an Fc polypeptide. In some cases, each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence, shown in FIG. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence shown in SEQ ID NO: 213, with amino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg , His, Asp or Glu. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide containing in the direction from the N-terminus to the C-terminus: i) the first variant of the 4-1BBL polypeptide of the present invention; ii) a second variant of the 4-1BBL polypeptide of the present invention; and iii) a third variant of the 4-1BBL polypeptide of the present invention; iv) MHC class I heavy chain; and v) an Fc polypeptide. In some cases, each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence, shown in FIG. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence shown in SEQ ID NO: 213, with amino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg , His, Asp or Glu. In some cases, the multimeric polypeptide of the present invention contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; and ii) a b2M polypeptide; and b) a second polypeptide containing in the direction from the N-terminus to the C-terminus: i) the first variant of the 4-1BBL polypeptide of the present invention; ii) a linker; iii) a second variant of the 4-1BBL polypeptide of the present invention; iv) a linker; v) a third variant of the 4-1BBL polypeptide of the present invention; vi) MHC class I heavy chain; and vii) an Fc polypeptide. In some cases, each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% relative to the amino acid sequence, shown in FIG. 36D, where amino acid 227 (denoted x) is an amino acid other than glutamine, e.g., where amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; or each of the first, second and third variants of the 4-1BBL polypeptides contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least 99% with the amino acid sequence shown in SEQ ID NO: 213, with amino acid substitution at Q147, e.g., where amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg , His, Asp or Glu. In some cases, the linker contains the sequence (GSSSS)n (SEQ ID NO: 131), where n is 1, 2, 3, 4, or 5. In some cases, the linker contains the sequence (GSSSS)n (SEQ ID NO: 131), where n is 4. In some cases, the linker contains the sequence (GSSSS)n (SEQ ID NO: 131), where n is 5. In some cases, the linker contains the sequence (GGGGS) _n , where n is 1, 2, 3, 4, 5 , 6, 7, 8, 9, or 10. In some cases, the linker contains the sequence (GGGGS) _n , where n is 2. In some cases, the linker contains the sequence (GGGGS) _n , where n is 3. In some cases, the linker contains the sequence ( GGGGS) _n , where n is 4. In some cases, the linker contains the sequence (GGGGS) _n , where n is 5.

Several immunomodulatory domains.

As noted above, in some cases, the multimeric polypeptide contains two or more immunomodulatory polypeptides. In some instances, at least one of the two or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide. For example, in the case of IL-2/synTac, in some instances, at least one of the two or more immunomodulatory polypeptides is a variant IL-2 polypeptide. As another example, in the case of 4-1BBL/synTac, in some instances at least one of the two or more immunomodulatory polypeptides is a variant of the 4-1BBL polypeptide.

In some cases, the multimeric polypeptide contains two or more copies of the variant IL-2 polypeptide of the present invention. In some instances, two or more IL-2 polypeptide variants are in the same polypeptide chain of a multimeric polypeptide. In some cases, two or more variants of the IL-2 polypeptides are in separate polypeptide chains of the multimeric polypeptide.

In some instances, the multimeric polypeptide comprises a first immunomodulatory polypeptide and at least a second immunomodulatory polypeptide, wherein the first immunomodulatory polypeptide is a variant IL-2 polypeptide of the present invention and the second immunomodulatory polypeptide is not an IL-2 polypeptide. For example, in some instances, the second immunomodulatory polypeptide is a member of the tumor necrosis factor (TNF) superfamily; for example, FasL polypeptide, 4-1BBL polypeptide, CD40 polypeptide, OX40L polypeptide, CD30L polypeptide, CD70 polypeptide, etc. In some instances, the second immunomodulatory polypeptide of the multimeric polypeptide is a T cell costimulatory polypeptide and is a member of the immunoglobulin (Ig) superfamily; for example, a CD7 polypeptide, a CD86 polypeptide, an ICAM polypeptide, etc. In some cases, the second immunomodulatory polypeptide is 4-1BBL, OX40L, ICOSL, ICAM, PD-L1, CD86, FasL and PD-L2. Suitable immunomodulatory polypeptides of the multimeric polypeptide of the present invention include, for example, CD7, CD30L, CD40, CD70, CD83, HLAG, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4, or HVEM.

Additional T cell modulating domains (MODs) that may be included in the multimeric polypeptide of the present invention include naturally occurring or synthetic human gene products (protein), affinity reagents (e.g., antibody, antibody fragment, single chain Fvs, aptamers, nanobody ) that target the human gene product, including, but not limited to, all secreted proteins resulting from classical and non-classical (e.g., FGF2, IL1, S100A4) secretion mechanisms, and the ecto-domains of all cell surface proteins anchored through natural genetics. encoded protein segments (monotopic or polytopic membrane) or post-translational modifications such as GPI linkages). Any natural or synthetic affinity reagent (eg, antibody, antibody fragment, single chain Fv, aptamer, nanobody, lectin, etc.) targets a cell surface glycan or other post-translational modification (eg, sulfated sites). Examples include, but are not limited to, TNF/TNFR family members (OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70) or affinity reagents directed at members of the TNF/TNFR family; members of the immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T-cell receptor chains, ICOS, ICOS ligand, HHLA2, butyrophilins, BTLA, B7 -H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR), killer cell immunoglobulin-like receptors (KIR)) , members of the lectin superfamily, selectins, cytokine/chemokine and cytokine/chemokine receptors, growth factors and growth factor receptors, adhesion molecules (integrins, fibronectins, cadherins) or polytopic integral membrane protein ectodomains or affinity reagents directed to the immunoglobulin superfamily and listed gene products . As well as active homologs/orthologs of such gene products, including but not limited to viral sequences (e.g. CMV, EBV), bacterial sequences, fungal sequences, eukaryotic pathogens (e.g. Schistosoma, Plasmodium, Babesia, Eimeria, Theileria, Toxoplasma) , Entamoeba, Leishmania, and Trypanosoma) and mammalian-derived coding regions. In addition, the MOD may contain a small molecule drug targeting a human gene product.

additional polypeptides.

The polypeptide chain of a multimeric polypeptide may include one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. One or more additional polypeptides may be included at the N-terminus of the multimeric polypeptide polypeptide chain, at the C-terminus of the multimeric polypeptide polypeptide chain, or within the multimeric polypeptide polypeptide chain.

epitope marks.

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 79); FLAG (e.g., DYKDDDDK (SEQ ID NO: 80); c-myc (e.g., EQKLISEEDL; SEQ ID NO) :81) and the like.

affine domains.

Affinity domains include peptide sequences that can interact with a binding partner, such as one that is immobilized on a solid support, such a partner is useful for identification and purification. DNA sequences encoding multiple consecutive single-stranded amino acids, such as histidine, when condensed to an expressed protein can be used to perform a one-step purification of a recombinant protein by high affinity binding to a resin column such as nickel sepharose. Examples of affine domains include

His5 (HHHH) (SEQ ID NO:82),

HisX6 (HHHHH) (SEQ ID NO:83), C-myc (EQKLISEEDL) (SEQ ID NO:81), Flag (DYKDDDDK) (SEQ ID NO:80), StrepTag (WSHPQFEK) (SEQ ID NO:84), hemagglutinin, e.g. HA tag (YPYDVPDYA; SEQ ID NO:79), glutathino n-S-transferase (GST), thioredoxin, cellulose binding domain,

RYIRS (SEQ ID NO:85), Phe-His-His-Thr (SEQ ID NO:86), chitin-binding domain, S-peptide, T7-peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA ( SEQ ID NO: 87), metal binding domains, e.g. zinc binding domains or calcium binding domains, such as those derived from calcium binding proteins, e.g. calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin , VILIP, neurocalcin, hippocalcin, friquenin, centrin, calpain large subunits, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K and calretinin, inteins, biotin, streptavidin, MyoD, halibut sequences, and maltose-binding protein.

Examples of IL-2/multimeric polypeptides.

The following are non-limiting embodiments of a multimeric IL2/synTac polypeptide suitable for use in the method of treatment of the present invention.

In some cases, the multimeric IL-2/synTac polypeptide contains: a) a first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a b2-microglobulin (b2M) polypeptide containing the amino acid sequence shown in FIG. 34A; and b) a second polypeptide comprising in N-terminal to C-terminal direction: i) an IL-2 polypeptide variant of the present invention; ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. In some instances, the variant IL-2 polypeptide contains the H16A and F42A substitutions. In some cases, the IgG1 Fc polypeptide contains the N297A substitution. In some cases, the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution. In some cases, the IgG1 Fc polypeptide contains an L234F substitution and an L235E substitution. In some cases, the IgG1 Fc polypeptide contains an L234F substitution, an L235E substitution, and a P331S substitution. In some instances, the second polypeptide contains two copies of a variant IL-2 polypeptide. In some instances, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In some instances, the second polypeptide comprises a peptide linker between one or more of: a) a first copy of the variant IL-2 polypeptide and a second copy of the variant IL-2 polypeptide; b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 207), (GGGGS) 4 (SEQ ID NO: 208) and AAAGG (SEQ ID NO: 73). In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33V. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33d.

In some cases, the multimeric polypeptide contains: a) the first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope; ii) a β2-microglobulin polypeptide comprising the amino acid sequence shown in FIG. 34A; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) an IL-2 polypeptide variant comprising the amino acid sequence shown in FIG. 34V; ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. In some cases, the IgG1 Fc polypeptide contains the N297A substitution. In some cases, the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution. In some cases, the IgG1 Fc polypeptide contains an L234F substitution and an L235E substitution. In some cases, the IgG1 Fc polypeptide contains an L234F substitution, an L235E substitution, and a P331S substitution. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33V. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33d. In some instances, the second polypeptide contains two copies of a variant IL-2 polypeptide. In some instances, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In some instances, the second polypeptide contains a peptide linker between one or more of: a) a first copy of the variant IL-2 polypeptide and a second copy of the variant IL-2 polypeptide;

b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS)3 (SEQ

ID NO: 207), (GGGGS)4 (SEQ ID NO: 208) and AAAGG (SEQ ID NO: 73).

In some cases, the multimeric polypeptide contains: a) a first polypeptide containing in the direction from the N-terminus to the C-terminus: i) an epitope containing the amino acid sequence YMLDLQPETT (SEQ ID NO. 77); v) a 32-microglobulin polypeptide containing the amino acid sequence shown in FIG. 34A; and b) a second polypeptide comprising, from the N-terminus to the C-terminus: i) an IL-2 polypeptide variant comprising the amino acid sequence shown in FIG. 34V; ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising the amino acid sequence depicted in FIG. 33A, 33B, 33C or 33D. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33V. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33d. In some instances, the second polypeptide contains two copies of a variant IL-2 polypeptide. In some instances, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In some instances, the second polypeptide comprises a peptide linker between one or more of: a) a first copy of the variant IL-2 polypeptide and a second copy of the variant IL-2 polypeptide; b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide. In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 207), (GGGGS) 4 (SEQ ID NO: 208) and AAAGG (SEQ ID NO: 73). In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33V. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33d.

In some cases, the multimeric polypeptide contains:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent shown in FIG. 22.

In some cases, the multimeric polypeptide contains:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent shown in FIG. 25.

In some cases, the multimeric polypeptide contains:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent shown in FIG. 28.

Compositions, doses and routes of administration.

In carrying out the treatment method of the present invention, synTac may be formulated in a composition containing a pharmaceutically acceptable excipient, and the immune checkpoint inhibitor may be formulated in a composition containing a pharmaceutically acceptable excipient. For simplicity, the term active ingredient is used below to refer to synTac or an immune checkpoint inhibitor. In general, synTac and the immune checkpoint inhibitor are present in separate compositions.

The composition may contain a pharmaceutically acceptable excipient, a wide variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been described in detail in various publications including, for example, Remington: The Science and Practice of Pharmacy, 19 ^th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, ^20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., ^3rd ed. amer. Pharmaceutical Assoc.

The pharmaceutical composition may contain synTac or an immune checkpoint inhibitor and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition will be suitable for administration to a subject, for example, will be sterile. For example, in some instances, a pharmaceutical composition will be suitable for human administration, such as when the composition is sterile and free of detectable pyrogens and/or other toxins.

The compositions may include other components such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. pharmaceutical quality. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions such as pH adjusting agents and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, lactate. sodium, hydrochlorides, sulfates, solvates (eg mixed ionic salts, water, organics), hydrates (eg water) and the like.

For example, compositions may include aqueous solution, powder form, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition may be formulated according to the various routes of administration described below.

When the active ingredient (synTac or immune checkpoint inhibitor) is injected (eg, subcutaneously, intraperitoneally, intramuscularly, and/or intravenously) directly into tissue, the formulation may be provided as a ready-to-use dosage form or as a non-aqueous form (eg. , a reconstituted storage stable powder) or in an aqueous form, such as a liquid, consisting of pharmaceutically acceptable carriers and excipients. The compositions can also be provided with the ability to increase the serum half-life of the active substance after administration. For example, the protein may be presented as a liposomal composition formulated as a colloid or according to other conventional methods to increase serum half-life. Many methods are available for preparing liposomes, as described, for example, in Szoka et al., 1980 Ann. Rev. Biophys. Bioeng. 9467, US Pat.

Other examples of formulations suitable for parenteral administration include isotonic sterile injectables, antioxidants, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickeners, stabilizers and preservatives. For example, the pharmaceutical composition in question may be present in a container, such as a sterile container such as a syringe. The compositions may be presented in sealed single or multiple dose containers, such as ampoules and vials, and may be stored under freeze-dried conditions (lyophilized conditions) requiring only the addition of a sterile liquid vehicle, such as water for injection, immediately prior to use. Injectable solutions and suspensions for immediate administration may be prepared from sterile powders, granules and tablets.

The concentration of active ingredient (synTac or immune checkpoint inhibitor) in a formulation can vary widely (e.g., from less than about 0.1%, typically 2% or at least about 2%, to 20% to 50% or more according to mass) and is usually selected based primarily on fluid volumes, viscosity, and patient-related factors, according to the particular route of administration chosen and the needs of the patient.

The present disclosure provides a container containing an active agent (synTac or an immune checkpoint inhibitor), such as a container containing a liquid formulation containing the active agent. The container may be, for example, a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, the container and composition are sterile.

The present disclosure relates to compositions, including pharmaceutical compositions, containing the active substance (synTac or immune checkpoint inhibitor). The composition may contain:

a) the active substance (synTac or immune checkpoint inhibitor); And

b) filler, as described above.

In some embodiments, the excipient is a pharmaceutically acceptable excipient.

Compositions containing a nucleic acid or a recombinant expression vector.

In some cases, synTac is administered as a multimeric polypeptide as such. In other cases, instead of introducing synTac as a multimeric polypeptide as such, one or more nucleic acids containing nucleotide sequences encoding synTac are introduced. Nucleic acids may be present in the pharmaceutical composition. The pharmaceutical composition may contain one or more recombinant expression vectors containing one or more nucleic acids. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been described in detail in various publications including, for example, A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, ^20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C Ansel et al., eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., ^3rd ed. amer. Pharmaceutical Assoc.

The pharmaceutical composition may include a nucleic acid or recombinant expression vector of the present invention in an amount of from about 0.001% to about 90% w/w. In the description of the formulations below, the term nucleic acid or recombinant expression vector will be

- 106 042111 be understood as including a nucleic acid or recombinant expression vector containing nucleotide sequences encoding synTac.

The nucleic acid or recombinant expression vector may be mixed, encapsulated, conjugated, or otherwise associated with other compounds or mixtures of compounds; such compounds may include, for example, liposomes or receptor molecules. The nucleic acid or recombinant expression vector can be formulated with one or more components that promote uptake, distribution and/or absorption.

The composition of the nucleic acid or recombinant expression vector can be formulated into any of a variety of possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The composition of the nucleic acid or recombinant expression vector can also be formulated as a suspension in an aqueous, non-aqueous or mixed medium. Aqueous suspensions may additionally contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

The composition containing the nucleic acid or recombinant expression vector may be a liposome composition. As used herein, the term liposome means a vesicle composed of amphiphilic lipids arranged in a bilayered sphere or bilayer. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the delivery composition. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH sensitive or negatively charged are believed to trap DNA rather than form complexes with it. Both cationic and non-cationic liposomes can be used to deliver the nucleic acid or recombinant expression vector in question.

Liposomes also include sterically stabilized liposomes, a term which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in increased circulation time compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers such as a polyethylene glycol (PEG) moiety. Liposomes and their use are further described in US Pat. No. 6287860, incorporated herein in its entirety by reference.

Compositions and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and emulsions is well known in the art. Surfactants and their use are further described in US Pat. No. 6287860.

In one embodiment, various penetration enhancers are included for efficient delivery of nucleic acids. In addition to facilitating the diffusion of non-lipophilic drugs across cell membranes, permeation enhancers also increase the permeability of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad categories, ie. surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants. Penetration enhancers and their use are further described in US Pat. No. 6,287,860, incorporated herein in its entirety by reference.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickening agents, flavoring agents, diluents, emulsifiers, dispersants or binders may be desirable. Suitable oral formulations include those in which said antisense nucleic acid is administered in combination with one or more penetration enhancers, surfactants, and chelators. Suitable surfactants include, but are not limited to, fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts and fatty acids and their use are further described in US Pat. No. 6,287,860. Also suitable are combinations of penetration enhancers, eg fatty acids/salts in combination with bile acids/salts. An example of a suitable combination is the sodium salt of lauric acid, capric acid and UDCA. Other penetration enhancers include, but are not limited to, fulloxyethylene 9-lauryl ether and fulloxyethylene 20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanoamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol, and AZONE™.

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Methods of treatment.

The present disclosure provides a method of treatment comprising administering synTac and an immune checkpoint inhibitor. In some cases, the method includes administering to an individual in need thereof:

a) the first composition containing synTac; And

b) a second composition containing an immune checkpoint inhibitor.

In some instances, the method of treatment of the present invention comprises administering to an individual in need thereof:

a) the first composition containing one or more recombinant expression vectors containing nucleotide sequences encoding synTac; And

b) a second composition containing an immune checkpoint inhibitor.

In some instances, the method of treatment of the present invention comprises administering to an individual in need thereof:

a) a first composition containing one or more mRNA molecules containing nucleotide sequences encoding a multimeric polypeptide; And

b) a second composition containing an immune checkpoint inhibitor.

In some instances, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint polypeptide.

Thus, for example, the method of treatment of the present invention may include the co-administration of synTac (eg, synTac 4-1BBL, synTac IL-2, etc. as described above) and an immune checkpoint specific antibody. By co-administration is meant that both synTac (e.g., synTac 4-1BBL, synTac IL-2, etc., as described above) and the checkpoint-specific antibody are administered to the individual, although not necessarily at the same time. the same time, to achieve a therapeutic effect that results from the use of both synTac and an immune checkpoint inhibitor. Administration of synTac (e.g., synTac 4-1BBL, synTac IL-2, etc.) and immune checkpoint specific antibody can be substantially simultaneous, e.g., synTac (e.g., synTac 4-1BBL, IL-2 synTac etc.) may be administered to an individual over about 1 minute to about 24 hours (e.g., about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, for about 4 hours, for about 8 hours, for about 12 hours, or for about 24 hours) relative to the introduction of an antibody specific to the immune checkpoint. In some instances, synTac (eg, synTac 4-1BBL, synTac IL-2, etc.) is administered to an individual who is being treated with an antibody specific for an immune checkpoint. Administration of synTac (eg, synTac 4-1BBL, synTac IL-2, etc.) and immune checkpoint specific antibody may occur at different times and/or frequencies.

Thus, for example, the method of treatment of the present invention may include the co-administration of synTac 4-1BBL and an antibody specific for an immune checkpoint. By co-administration is meant that the individual is administered synTac 4-1BBL and a checkpoint-specific antibody, although not necessarily at the same time, to achieve a therapeutic effect that results from the use of both synTac and an immune checkpoint inhibitor. Administration of synTac 4-1BBL and the immune checkpoint specific antibody can be substantially simultaneous, e.g., synTac 4-1BBL can be administered to an individual over a period of about 1 minute to about 24 hours for about 5 minutes, for about 15 minutes, for about 30 minutes, for about 1 hour, for about 4 hours, for about 8 hours, for about 12 hours, or for about 24 hours) relative to administration of the antibody specific to the immune checkpoint. In some cases, synTac 4-1BBL is administered to an individual who is being treated with an antibody specific for an immune checkpoint. Administration of synTac 4-1BBL and immune checkpoint specific antibody may occur at different times and/or frequencies.

As another example, the method of treatment of the present invention may include the co-administration of synTac (eg, synTac IL-2, as described above) and an antibody specific for an immune checkpoint. By co-administration is meant that the individual is administered synTac IL-2 and a checkpoint-specific antibody, although not necessarily at the same time, to achieve a therapeutic effect that results from both synTac and an immune checkpoint inhibitor. Administration of synTac IL-2 and immune checkpoint specific antibody can be substantially simultaneous, e.g., synTac IL-2 can be administered to an individual over a period of about 1 minute to about 24 hours for about 5 minutes, for about 15 minutes, for about 30 minutes, for about 1 hour, for about 4 hours, for about 8 hours, for about 12 hours, or for about 24 hours) relative to administration of the antibody specific to the immune checkpoint. In some cases, synTac IL-2 is administered to an individual who is being treated with an antibody specific for an immune checkpoint. Administration of synTac IL-2 and immune checkpoint specific antibody may occur at different times and/or frequencies.

The present disclosure provides a method of treatment comprising administering synTac and an immune checkpoint inhibitor. The method of treatment of the present invention can modulate the activity of a target T cell. In some cases, for example, when the target T cell is a CD8+ T cell, the multimeric polypeptide contains MHC class I polypeptides (eg, β2-microglobulin and MHC class I heavy chain). In some cases, for example, when the target T cell is a CD4+ T cell, the multimeric polypeptide contains MHC class II polypeptides (eg, MHC class II α-chain; MHC class II β-chain).

When the multimeric polypeptide comprises an immunomodulatory polypeptide that is an activating polypeptide, the method of the present invention activates an epitope-specific T cell. In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present in a cancer cell, and contact of the epitope-specific T cell with a multimeric polypeptide increases the cytotoxic activity of the T cell against the cancer cell. In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present in a cancer cell, and the method of the present invention increases the number of epitope-specific T cells.

In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the cytotoxic activity of the T cell against the virus-infected cell. In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present in a virus-infected cell, and the method of the present invention increases the number of epitope-specific T cells.

When the multimeric polypeptide of the present invention comprises an immunomodulatory polypeptide that is an inhibitory polypeptide, the method of the present invention inhibits an epitope-specific T cell. In some instances, the epitope-specific T cell is a self-reactive T cell that is specific for an epitope present on the self-antigen, and the method of the present invention reduces the number of self-reactive T cells.

In some instances, the immunomodulatory polypeptide is an activating polypeptide and the multimeric polypeptide activates an epitope-specific T cell. In some instances, the epitope is a cancer-associated epitope and the multimeric polypeptide increases the activity of a T cell specific for the cancer-associated epitope.

In some cases, the method of treatment of the present invention treats cancer in an individual having cancer. Thus, the present disclosure provides a method for treating cancer in an individual, the method comprising administering to the individual:

a) a multimeric polypeptide of the present invention, or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) containing nucleotide sequences encoding a multimeric polypeptide, wherein the multimeric polypeptide comprises a T cell epitope that is a cancer epitope, and where the multimeric polypeptide contains a stimulatory immunomodulatory polypeptide; And

b) immune checkpoint inhibitor.

In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts which, when administered in one or more doses to an individual in need thereof, reduce the number of cancer cells in the individual. For example, in some instances, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts which, when administered in one or more doses to an individual in need thereof, reduce the amount of cancer cells in the individual by at least 10%, at least at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least by 70%, at least 80%, at least 90%, or at least 95% compared to the number of cancer cells in the individual before administration of the multimeric polypeptide and immune checkpoint inhibitor, or in the absence of administration of the multimeric polypeptide and immune inhibitor checkpoint. In some instances, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce the amount of cancer cells in the individual to undetectable levels. In some instances, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts which, when administered in one or more doses to an individual in need thereof, reduce the tumor mass of the individual. For example, in some instances, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts that, when administered in one or more doses to an individual in need thereof, reduce the individual's tumor weight by at least 10%, by

- 109 042111 at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60% , at least 70%, at least 80%, at least 90%, or at least 95% of the individual's tumor weight before administration of the multimeric polypeptide and immune checkpoint inhibitor, or in the absence of administration of the multimeric polypeptide and an immune checkpoint inhibitor. In some cases, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts which, when administered in one or more doses to an individual in need thereof, increase the survival time of the individual. For example, in some instances, effective amounts of the multimeric polypeptide and the immune checkpoint inhibitor are amounts which, when administered in one or more doses to an individual in need thereof, increase the individual's survival time by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years compared to the individual's expected survival time in the absence of administration of a multimeric polypeptide and an immune checkpoint inhibitor.

In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present on a virus-infected cell, and the method of the present invention increases the cytotoxic activity of the T cell against the virus-infected cell. In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present in a virus-infected cell, and the method of the present invention increases the number of epitope-specific T cells.

As noted above, in some instances, in a method of treating a subject, a multimeric polypeptide is administered to an individual in need thereof as the polypeptide itself. In other instances, in carrying out a method of treating a subject, one or more nucleic acids containing nucleotide sequences encoding a multimeric polypeptide are administered to an individual in need thereof. Thus, in other cases, one or more nucleic acids encoding synTac are administered to an individual in need thereof.

Doses - synTac.

Suitable doses of synTac may be determined by the attending physician or other qualified medical personnel based on various clinical conditions. As is well known in medicine, dosages for any given patient depend on many factors, including patient size, body surface area, age, particular polypeptide or nucleic acid administered, gender of the patient, time and route of administration, general health, and other concomitantly administered drugs. The multimeric polypeptide (synTac) can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, for example, from 0.1 mg/kg body weight to 10 mg/kg body weight, for example, from 0.5 mg/kg body weight up to 5 mg/kg body weight; however, doses below and above this exemplary range are contemplated, especially given the above factors. If the regimen is a continuous infusion, doses may also be in the range of 1 µg to 10 mg per kilogram of body weight per minute. The multimeric polypeptide may be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, for example, from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg. kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg /kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight up to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of the multimeric polypeptide is 0.01 µg to 100 g per kg of body weight, 0.1 µg to 10 g per kg of body weight, 1 µg to 1 g per kg of body weight, 10 µg to 100 mg per kg of body weight, from 100 mcg to 10 mg per kg of body weight, or from 100 mcg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition rate for dosing based on the measured retention time and concentrations of the administered agent in body fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, in which the multimeric polypeptide is administered at maintenance doses in the range from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight body, from 1 µg to 1 g per kg of body weight, from 10 µg to 100 mg per kg of body weight, from 100 µg to 10 mg per kg of body weight, or from 100 µg to 1 mg per kg of body weight.

Those skilled in the art will appreciate that dosage levels may vary as a function of the particular multimeric polypeptide, the severity of symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of said compound by various means.

In some embodiments, multiple doses of a multimeric polypeptide (or nucleic acid or recombinant expression vector encoding them) are administered. The frequency of administration of the multimeric polypeptide may vary depending on any of a variety of factors,

- 110 042111 e.g. severity of symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The duration of administration of the multimeric polypeptide, eg, the period of time over which the multimeric polypeptide is administered, may vary depending on any of a variety of factors, such as patient response, etc.

For example, the multimeric polypeptide may be administered over a period of one day to one week, two weeks to four weeks, one month to two months, two months to four months, four months to six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.

Suitable doses of synTac may be determined by the attending physician or other qualified medical personnel based on various clinical conditions. As is well known in medicine, dosages for any given patient depend on many factors, including patient size, body surface area, age, particular polypeptide or nucleic acid administered, gender of the patient, time and route of administration, general health, and other concomitantly administered drugs. The multimeric polypeptide (synTac) can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, for example, from 0.1 mg/kg body weight to 10 mg/kg body weight, for example, from 0.5 mg/kg body weight up to 5 mg/kg body weight; however, doses below and above this exemplary range are contemplated, especially given the above factors. If the regimen is a continuous infusion, doses may also be in the range of 1 µg to 10 mg per kilogram of body weight per minute. The multimeric polypeptide may be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, for example, from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg. kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg /kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight up to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of the multimeric polypeptide is 0.01 µg to 100 g per kg of body weight, 0.1 µg to 10 g per kg of body weight, 1 µg to 1 g per kg of body weight, 10 µg to 100 mg per kg of body weight, 100 mcg to 10 mg per kg of body weight, or 100 mcg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition rate for dosing based on the measured retention time and concentrations of the administered agent in body fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, in which the multimeric polypeptide is administered at maintenance doses in the range from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight body, from 1 µg to 1 g per kg of body weight, from 10 µg to 100 mg per kg of body weight, from 100 µg to 10 mg per kg of body weight, or from 100 µg to 1 mg per kg of body weight.

Those skilled in the art will appreciate that dosage levels may vary as a function of the particular multimeric polypeptide, the severity of symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of said compound by various means.

In some embodiments, multiple doses of a multimeric polypeptide (or nucleic acid or recombinant expression vector encoding them) are administered. The frequency of administration of the multimeric polypeptide may vary depending on any of a variety of factors, such as the severity of symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The duration of administration of the multimeric polypeptide, such as the period of time over which the multimeric polypeptide is administered, may vary depending on any of a variety of factors, such as patient response, etc. For example, the multimeric polypeptide may be administered over a period of one day to one week, two weeks to four weeks, one month to two months, two months to four months, four months to six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.

Doses - immune checkpoint inhibitor.

Suitable doses of the immune checkpoint inhibitor can be determined by the attending physician or other qualified medical personnel based on various clinical indications. As is well known in medicine, dosages for any given patient depend on many factors, including patient size, body surface area, age, particular polypeptide or nucleic acid administered, patient sex, time and route of administration, general health, and other administered drugs at the same time. The immune checkpoint inhibitor can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight, for example, from 0.1 mg/kg body weight to 10 mg/kg body weight, for example, from 0.5 mg /kg of body weight up to 5 mg/kg of body weight; however, doses below and above this exemplary range are contemplated, especially given the above factors. If the regimen is a continuous infusion, doses may also be in the range of 1 µg to 10 mg per kilogram of body weight per minute. The immune checkpoint inhibitor may be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, for example, from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg . kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg /kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of an immune checkpoint inhibitor is 0.01 µg to 100 g per kg of body weight, 0.1 µg to 10 g per kg of body weight, 1 µg to 1 g per kg of body weight, 10 µg up to 100 mg per kg of body weight, from 100 mcg to 10 mg per kg of body weight, or from 100 mcg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition rate for dosing based on the measured retention time and concentrations of the administered agent in body fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, in which the immune checkpoint inhibitor is administered at maintenance doses in the range from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, 1 µg to 1 g per kg of body weight, 10 µg to 100 mg per kg of body weight, 100 µg to 10 mg per kg of body weight, or 100 µg to 1 mg per kg of body weight.

Those skilled in the art will appreciate that dosage levels may vary as a function of the particular immune checkpoint inhibitor, the severity of symptoms, and the subject's susceptibility to side effects. Those skilled in the art can readily determine preferred dosages of said compound by various means.

In some embodiments, multiple doses of an immune checkpoint inhibitor are administered. The frequency of administration of the immune checkpoint inhibitor may vary depending on any of a variety of factors such as severity of symptoms, etc. For example, in some embodiments, the multimeric polypeptide is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

The duration of administration of the immune checkpoint inhibitor, eg, the period of time for which the immune checkpoint inhibitor is administered, may vary depending on any of a variety of factors, such as patient response, etc. For example, the immune checkpoint inhibitor may be administered over a period of about one day to about one week, about two weeks to about four weeks, about one month to about two months, about two months to about four months. , from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.

The following are non-limiting examples.

Pembrolizumab may be administered to an individual in need at 2 mg/kg every 3 weeks. Pembrolizumab can be administered to an individual in need of it in an amount of 200 mg every 3 weeks. In some cases, the method of the present invention reduces the amount of anti-PD1 that must be entered to achieve a clinical effect. For example, in some cases, the amount of pembrolizumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of pembrolizumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Nivolumab can be administered to an individual in need at 3 mg/kg every 2 weeks. Nivolumab can be administered to an individual in need of it in an amount of 240 mg every 2 weeks. In some cases, the method of the present invention reduces the amount of anti-PDl that must be entered to achieve a clinical effect. For example, in some cases, the amount of nivolumab that needs to be administered to achieve a clinical effect can be reduced by 10-50% or more than 50% compared to the amount of nivolumab that needs to be administered to achieve a clinical effect in the absence of treatment with synTac.

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Atezolizumab may be administered to an individual in need of it in an amount of 1200 mg every 3 weeks. In some cases, the method of the present invention reduces the amount of anti-PDl that must be entered to achieve a clinical effect. For example, in some cases, the amount of atezolizumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of atezolizumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Ipilimumab can be administered to an individual in need at 3 mg/kg every 3 weeks. Ipilimumab can be administered to an individual in need at 10 mg/kg every 3 weeks. Ipilimumab may be administered to a subject in need at 10 mg/kg every 12 weeks. In some cases, the method of the present invention reduces the amount of anti-PDl that must be entered to achieve a clinical effect. For example, in some cases, the amount of ipilimumab that must be administered to achieve a clinical effect may be reduced by 10-50% or more than 50% compared to the amount of ipilimumab that must be administered to achieve a clinical effect in the absence of treatment with synTac.

Ways of introduction.

The active substance (multimeric polypeptide; immune checkpoint inhibitor) is administered to the individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration. In some cases, synTac is administered by the first route of administration; and the immune checkpoint inhibitor is administered by a second route of administration that is different from the first route of administration. In some cases, synTac and the immune checkpoint inhibitor are administered by the same routes of administration.

Common and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or adjusted as necessary, depending on the multimeric polypeptide, immune checkpoint inhibitor, and/or desired effect.

In some cases, the multimeric polypeptide is administered intravenously; and an immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered intramuscularly; and an immune checkpoint inhibitor is administered intramuscularly. In some cases, the multimeric polypeptide is administered topically; and an immune checkpoint inhibitor is administered topically. In some cases, the multimeric polypeptide is administered intratumorally; and an immune checkpoint inhibitor is administered intratumorally. In some cases, the multimeric polypeptide is administered peritumorally; and an immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intracranially; and an immune checkpoint inhibitor is administered intracranially. In some cases, the multimeric polypeptide is administered subcutaneously; and an immune checkpoint inhibitor is administered subcutaneously.

In some cases, the multimeric polypeptide is administered intravenously; and an immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intramuscularly; and an immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered systemically; and an immune checkpoint inhibitor is administered topically. In some cases, the multimeric polypeptide is administered intratumorally; and an immune checkpoint inhibitor is administered intravenously. In some cases, the multimeric polypeptide is administered systemically; and an immune checkpoint inhibitor is administered peritumorally. In some cases, the multimeric polypeptide is administered intravenously; and an immune checkpoint inhibitor is administered intracranially. In some cases, the multimeric polypeptide is administered subcutaneously; and an immune checkpoint inhibitor is administered intravenously.

The multimeric polypeptide and the immune checkpoint inhibitor may be administered to the host using any of the available conventional methods and routes suitable for conventional drug delivery, including systemic or local routes. In general, administration routes contemplated for use in the method of the present invention include, but are not limited to, enteral, parenteral, and inhalation routes.

Parenteral routes of administration other than inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intratumoral, intratumoral, peritumoral, and intravenous, i. any route of administration other than through the alimentary canal. Parenteral administration can be for systemic or local delivery of the active substance (synTac or immune checkpoint inhibitor). If systemic delivery is desired, administration may include intravenous delivery.

Suitable subjects for treatment.

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Subjects suitable for treatment with the method of the present invention include individuals who have cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have not responded to treatment, and individuals who have been treated for cancer and who initially showed a response to treatment but subsequently became refractory to treatment. Subjects suitable for treatment with the method of the present invention include individuals who have an infection (e.g., infection with a pathogen such as a bacterium, virus, protozoan, etc.), including individuals who have been diagnosed with an infection, and individuals who were treated for the infection but who did not respond to treatment. Subjects suitable for treatment with the method of the present invention include individuals who have a bacterial infection, including individuals who have been diagnosed with a bacterial infection, and individuals who have been treated for a bacterial infection but who have not shown a response to treatment. Subjects suitable for treatment with the method of the present invention include individuals who have a viral infection, including individuals who have been diagnosed with a viral infection, and individuals who have received treatment for a viral infection but who have not shown a response to treatment. Subjects suitable for treatment with the method of the present invention include individuals who have an autoimmune disease, including individuals diagnosed with an autoimmune disease, and individuals who have received treatment for an autoimmune disease but who have not shown a response to treatment.

In some instances, the method of the present invention comprises administering synTac to an individual who is being treated with an immune checkpoint inhibitor. In some instances, the method of the present invention comprises administering synTac to an individual who is being treated with an anti-PD1 antibody. For example, in some instances, the method of the present invention comprises administering synTac to an individual who is being treated with pembrolizumab. As another example, in some instances, the method of the present invention comprises administering synTac to an individual who is being treated with nivolumab. In some instances, the method of the present invention comprises administering synTac to an individual who is being treated with an anti-PD-L1 antibody. For example, in some instances, the method of the present invention comprises administering synTac to an individual who is being treated with atezolizumab. In some instances, the method of the present invention comprises administering synTac to an individual who is being treated with an anti-CTLA4 antibody. For example, in some instances, the method of the present invention comprises administering synTac to an individual who is being treated with ipilimumab. As another example, in some instances, the method of the present invention comprises administering synTac to an individual who is being treated with tremelimumab.

In some cases, for example, when the epitope is an HPV epitope, a subject suitable for treatment with the method of the present invention is an individual who has been diagnosed with an HPV-associated or HPV-associated cancer. HPV-associated and HPV-associated cancers include, for example, head and neck cancer; cervical cancer; and cancer of the genitourinary system.

Methods for selectively delivering to an individual a co-stimulatory polypeptide together with an immune checkpoint inhibitor.

The present disclosure thus provides a method for delivering a costimulatory polypeptide, such as IL-2, or a reduced affinity variant of a naturally occurring costimulatory polypeptide, such as an IL-2 variant disclosed herein, to a selected T cell or selected population of T cells. for example, in such a way that it targets the TCR specific for that epitope, together with the simultaneous administration of the checkpoint inhibitor, to provide the patient with a therapeutic effect of both the selective delivery of the co-stimulatory polypeptide and the checkpoint inhibitor. The present disclosure provides a method for delivering a costimulatory polypeptide, such as IL-2, or a reduced affinity variant of a naturally occurring costimulatory polypeptide, such as an IL-2 variant disclosed herein, selectively to a target T cell bearing a TCR specific for epitope present in the multimeric polypeptide of the present invention, together with the co-administration of a checkpoint inhibitor, to provide the patient with a therapeutic effect of both the selective delivery of the co-stimulatory polypeptide and the checkpoint inhibitor. The method includes contacting a population of T cells with a multimeric polypeptide of the present invention. The T cell population may be a mixed population that includes:

i) target T cell; and ii) non-target T cells that are not specific for an epitope (eg, T cells specific for epitopes other than the epitope to which the epitope-specific T cell binds).

The epitope-specific T cell is specific for an epitope-presenting peptide present in the multimeric polypeptide and binds to an HLA peptide complex or an MHC peptide complex provided by the multimeric polypeptide. Contacting a population of T cells with

The multimeric polypeptide delivers a costimulatory polypeptide (eg, IL-2 or an affinity reduced IL-2 variant) present in the multimeric polypeptide selectively to T cells that are specific for the epitope presented in the multimeric polypeptide. The checkpoint inhibitor is co-administered with the multimeric polypeptide (either together or at other times before and/or after the administration of the multimeric polypeptide) to provide the patient with the therapeutic effect of selectively delivering both the costimulatory polypeptide and the checkpoint inhibitor.

Thus, the present disclosure provides a method for delivering to a patient (i) a checkpoint inhibitor as described above and (ii) a co-stimulatory polypeptide such as IL-2 or a reduced affinity variant of a naturally occurring co-stimulatory polypeptide such as an IL variant. -2 disclosed herein, or a combination of both, selectively for a target T cell, the method comprising contacting a mixed population of T cells with a multimeric polypeptide of the present invention. The mixed T cell population includes target T cells and non-target T cells. The target T cell is specific for an epitope present in the multimeric polypeptide. Contacting a mixed population of T cells with a multimeric polypeptide of the present invention delivers the co-stimulatory polypeptide present in the multimeric polypeptide to the target T cell. Thus, co-administration of a checkpoint inhibitor with a multimeric polypeptide (either together or at other times before and/or after administration of the multimeric polypeptide) provides the patient with the therapeutic effect of selectively delivering both the co-stimulatory polypeptide and the checkpoint inhibitor.

For example, a multimeric polypeptide of the present invention is contacted with a population of T cells including:

i) target T cells that are specific for an epitope present in the multimeric polypeptide; and ii) non-target T cells, eg, T cells specific for a second epitope that is not an epitope present in the multimeric polypeptide.

Contact with a population results in selective delivery of a costimulatory polypeptide (e.g., a naturally occurring costimulatory polypeptide (e.g., naturally occurring IL-2) or a reduced affinity variant of a naturally occurring costimulatory polypeptide (e.g., an IL-2 variant disclosed herein) ), which is present in the multimeric polypeptide, to the target cell. Thus, for example, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 4%, 3%, 2% or 1% T non-target cells bind the multimeric polypeptide, and as a result, the costimulatory polypeptide (eg, IL-2 or IL-2 variant) present in the multimeric polypeptides is substantially not delivered to the non-target T cells. Thus, co-administration of a checkpoint inhibitor with a multimeric polypeptide (either together or at other times before and/or after administration of the multimeric polypeptide) provides the patient with the therapeutic effect of selectively delivering both the co-stimulatory polypeptide and the checkpoint inhibitor.

In some cases, the T cell population is an in vitro population. In some instances, the T cell population is an in vitro population and the biological response (e.g., activation and/or growth of T cells and/or phenotypic differentiation) of the target T cell population to the multimeric polypeptide of the present invention is elicited in the context of an in culture. vitro. For example, a mixed population of T cells may be obtained from an individual and may be contacted with the multimeric polypeptide in vitro. Such contact may include single or repeated exposure of populations of T cells to certain doses or modes of exposure. In some instances, said contacting results in selective binding/activation and/or growth of target T cells in a population of T cells and results in a population of activated and/or growing target T cells. As an example, a mixed population of T cells may be peripheral blood mononuclear cells (PBMCs). For example, a patient's PBMC can be obtained by standard methods of blood sampling and enrichment of PBMC prior to exposure to 0.1-1000 nM of the multimeric polypeptide of the present invention under standard lymphocyte culture conditions. At time points before, during and after exposure to a mixed population of T cells at a specific dose and schedule, the abundance of target T cells in in vitro culture can be monitored using specific peptide-MHC multimers and/or phenotypic markers and/or functional activity. (eg ELISpot assay for cytokines). In some cases, after reaching the optimal number and/or phenotype of antigen-specific cells in vitro, the entire population or part of the population of activated and/or growing target T cells is administered to an individual (an individual from whom a mixed population of T cells was taken) who already has been administered a checkpoint inhibitor prior to administration of target T cells and/or to which a checkpoint inhibitor will be administered after administration of target T cells.

In some cases, the T cell population is an in vitro population. For example, a mixed population of T cells is obtained from an individual and contacted with a multimeric polypeptide of the present invention in vitro. Such contact, which may include a single or

- 115 042111 repeated exposure of T cells with a certain dose and/or schedule of exposure in the context of cell culture in vitro, can be used to determine whether a mixed population of T cells includes specific to the epitope presented on the multimeric T cell polypeptide. The presentation of epitope-specific T cells of a multimeric polypeptide can be determined by analyzing a sample containing a mixed population of T cells, which population of T cells contains T cells that are not specific to the epitope (non-target T cells) and may contain T cells epitope-specific (T-cell targets). Known assays can be used to detect activation and/or proliferation of target T cells, thereby providing an ex vivo assay that can determine whether a particular multimeric polypeptide (synTac) possesses an epitope that binds to T cells presented in an individual, and thus whether the multimeric polypeptide has potential use as a therapeutic composition for that individual. Suitable known assays for detecting target T cell activation and/or proliferation include, for example, flow cytometric characterization of T cell phenotype and/or antigen specificity and/or proliferation. Such an assay to detect the presence of epitope-specific T cells, e.g., co-diagnosis, may further include additional assays (e.g., ELISpot assays for effector cytokines) and/or appropriate controls (e.g., antigen-specific and antigen-specific staining reagents for multimeric peptide-HLA) for determining whether the multimeric polypeptide selectively binds/activates and/or promotes the growth of target T cells. Thus, for example, the present disclosure provides a method for detecting in a mixed population of T cells derived from an individual a target T cell presentation that binds an epitope of interest, the method comprising:

a) contacting in vitro a mixed population of T cells with a multimeric polypeptide of the present invention, where the multimeric polypeptide contains the epitope under investigation; And

b) detecting T cell activation and/or proliferation in response to said contact, wherein activated and/or proliferated T cells indicate the presence of a target T cell.

Alternatively and/or additionally, if activation and/or growth (proliferation) of the desired T cell population is obtained using a multimeric polypeptide, then all or part of the T cell population, including activated/growing T cells, can be administered back to the individual (who has been administered checkpoint inhibitor prior to T cell administration and/or a checkpoint inhibitor will be administered after T cell administration) as therapy.

In some instances, the T cell population is an in vivo population in an individual. In such cases, the method of the present invention for selectively delivering a costimulatory polypeptide (eg, IL-2 or IL-2 with reduced affinity) to an epitope-specific T cell comprises administering the multimeric polypeptide to the individual.

An epitope-specific T cell into which a co-stimulatory polypeptide (eg, IL-2 or affinity reduced IL-2) is selectively delivered is also referred to herein as a target T cell. In some instances, the target T cell is a regulatory T cell (Treg). In some cases, Treg inhibits or suppresses the activity of an autoreactive T cell.

In some cases, the target T cell is a cytotoxic T cell. For example, the target T cell may be a cytotoxic T cell specific for a cancer epitope c (eg, an epitope presented by a cancer cell).

Examples of non-limiting aspects of the invention.

Aspects, including embodiments, of the proposed subject matter described above may provide an advantage alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, some non-limiting aspects of the disclosure are provided below, referred to as Aspects 1-122, Aspects A-Z, Aspects AA-ZZ, and Aspects AAA-BBB. Upon reading this description, those skilled in the art will appreciate that each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide the basis for all such aspect combinations and is not limited to the aspect combinations explicitly provided below.

Aspect 1 An IL-2 polypeptide variant comprising an amino acid sequence having at least 85% amino acid sequence identity as specified in SEQ ID NO: 1, wherein the IL-2 polypeptide variant has one or more amino acid substitutions relative to the sequence shown in SEQ ID NO: 1, and wherein the IL-2 polypeptide variant exhibits reduced binding affinity for the IL-2 receptor (IL2R) containing alpha, beta, and gamma polypeptides having the amino acid sequences depicted in FIG. 3A-3C compared to the binding affinity of the IL-2 amino acid sequence indicated in one of SEQ ID NO: 1 for IL2R.

Aspect 2. An IL2 polypeptide variant of Aspect 1, wherein the variant comprises substituting one or more of E15, H16, D20, F42, Y45, and Q126.

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Aspect 3 A variant IL2 polypeptide according to Aspect 1 or Aspect 2, wherein the variant immunomodulatory polypeptide exhibits less than 10% to less than 50% of the binding affinity exhibited by the IL2 amino acid sequence shown in SEQ ID NO: 1 for IL2R.

Aspect 4. An IL2 polypeptide variant according to any one of aspects 1-3, wherein the variant comprises substitutions of F42 with Ala, Gly, Val, Ile, or Leu.

Aspect 5 An IL2 polypeptide variant according to any one of Aspects 1-3, wherein the variant comprises F42 and D20 substitutions.

Aspect 6 An IL2 polypeptide variant according to any one of Aspects 1-3, wherein the variant comprises F42 and H16 substitutions.

Aspect 7. An IL2 polypeptide variant according to any one of aspects 1-3, wherein the variant comprises F42, D20 and Y45 substitutions; or where the variant includes replacements F42, H16 and Q126.

Aspect 8. A multimeric polypeptide comprising:

a) a first polypeptide containing, in order from N-terminus to C-terminus: i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory domains, wherein the one or more immunomodulatory domains are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide, wherein at least one of one or more immunomodulatory domains is a variant IL2 polypeptide according to any of aspects 1-7, and wherein the multimeric polypeptide exhibits reduced receptor binding affinity IL-2 (IL2R) containing alpha, beta and gamma polypeptides having the amino acid sequences shown in FIG. 3A-3C compared to the binding affinity of a control multimeric polypeptide containing the IL2 amino acid sequence shown in SEQ ID NO: 1 for the IL2R polypeptide.

Aspect 9. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide contains, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an IL2 polypeptide variant; And

b) the second polypeptide contains, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect 10. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide contains, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) the second polypeptide contains, in order from N-terminus to C-terminus:

i) an IL2 polypeptide variant;

ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide.

Aspect 11. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide contains, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) the second polypeptide contains, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an IL2 polypeptide variant.

Aspect 12. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide contains, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing in the direction from the N-terminus to the C-terminus:

i) an IL2 polypeptide variant; and ii) a second MHC polypeptide.

Aspect 13. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide contains, in order from N-terminus to C-terminus:

i) an epitope;

- 117 042111 ii) the first MHC polypeptide; and iii) an IL2 polypeptide variant; And

b) the second polypeptide contains a second MHC polypeptide.

Aspect 14 The multimeric polypeptide of aspect 8 wherein the non-Ig backbone is an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

Aspect 15. The multimeric polypeptide of any one of aspects 8-14, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide.

Aspect 16 The multimeric polypeptide of aspect 15, wherein the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 6.

Aspect 17 The multimeric polypeptide of aspect 15 wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B or HLA-C heavy chain.

Aspect 18 The multimeric polypeptide of aspect 15 wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 5A-5C.

Aspect 19. The multimeric polypeptide of any one of aspects 8-14, wherein the first MHC polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect 20 The multimeric polypeptide of any one of aspects 8-19 wherein the epitope is a T cell epitope.

Aspect 21. The multimeric polypeptide of any one of aspects 8-13 and 15-20, wherein the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or Fc IgM polypeptide.

Aspect 22 The multimeric polypeptide of Aspect 21 wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence shown in FIG. 4A-4C.

Aspect 23 The multimeric polypeptide of any one of aspects 8-22, wherein the first polypeptide and the second polypeptide are non-covalently linked.

Aspect 24 A multimeric polypeptide according to any one of aspects 8-22, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

Aspect 25 The multimeric polypeptide of aspect 24 wherein the covalent bond is through a disulfide bond.

Aspect 26. The multimeric polypeptide of claim 25, wherein the first MHC polypeptide or linker between the epitope and the first MHC polypeptide contains an amino acid substitution to obtain a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to obtain a second Cys residue, and wherein a disulfide bond is between the first and second residues of Cys.

Aspect 27. A multimeric polypeptide according to any one of aspects 8-26, comprising a linker located between the epitope and the first MHC polypeptide.

Aspect 28. A multimeric polypeptide according to any one of aspects 8-26, comprising a linker located between the MHC polypeptide and the immunomodulatory polypeptide.

Aspect 29 A multimeric polypeptide according to any one of aspects 8-28, comprising 2 variants of an IL2 polypeptide.

Aspect 30 A multimeric polypeptide according to any one of aspects 8-28, comprising 3 variants of an IL2 polypeptide.

Aspect 31 The multimeric polypeptide of Aspect 29 or Aspect 30, wherein 2 or 3 IL2 polypeptide variants are in tandem, and wherein the multimeric polypeptide contains a linker between the IL2 polypeptide variants.

Aspect 32. The multimeric polypeptide of any one of aspects 8-28, wherein the IL2 variant comprises a substitution of one or more of E15, H16, D20, F42, Y45, and Q126

Aspect 33 The multimeric polypeptide of any one of aspects 8-28, wherein the IL2 variant comprises replacing F42 with Ala, Gly, Val, Ile, or Leu.

Aspect 34 The multimeric polypeptide of aspect 33 wherein the IL2 variant contains F42 and D20 substitutions.

Aspect 35 The multimeric polypeptide of aspect 33 wherein the IL2 variant contains F42 and H16 substitutions.

Aspect 36. The multimeric polypeptide of aspect 33 wherein the IL2 variant comprises F42, D20 and Y45 substitutions; or where the variant IL-2 includes substitutions F42, H16 and Q126.

Aspect 37. Nucleic acid containing a nucleotide sequence encoding a polypeptide,

i) where the recombinant polypeptide contains, in order from N-terminus to C-terminus:

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a) an epitope;

b) the first major histocompatibility complex (MHC) polypeptide;

c) an immunomodulatory polypeptide;

d) a proteolytically cleavable linker or ribosome skipping signal;

e) a second MHC polypeptide; and

f) an immunoglobulin (Ig) Fc polypeptide;

where the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide according to any of aspects 1-7; or ii) wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus:

a) an epitope;

b) the first MHC polypeptide;

c) a proteolytically cleavable linker or ribosome skipping signal;

d) an immunomodulatory polypeptide;

e) a second MHC polypeptide; and

f) an Ig Fc polypeptide, wherein the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide according to any one of aspects 1-7.

Aspect 38. The nucleic acid of aspect 37, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide.

Aspect 39 The nucleic acid of aspect 38, wherein the p2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 6.

Aspect 40. The nucleic acid of aspect 38, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect 41 The nucleic acid of aspect 40, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 5A-5C.

Aspect 42. The nucleic acid of aspect 37, wherein the first MHC polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect 43 The nucleic acid of any one of aspects 37-43 wherein the epitope is a T cell epitope.

Aspect 44. The nucleic acid of any one of aspects 37-43, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

Aspect 45 The nucleic acid of Aspect 44, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence shown in FIG. 4A-4C.

Aspect 46 The nucleic acid of any one of aspects 37-45, wherein the IL2 immunomodulatory polypeptide variant comprises a substitution of one or more of E15, H16, D20, F42, Y45, and Q126.

Aspect 47. Nucleic acid according to any one of aspects 37-46, wherein the multimeric polypeptide comprises a second immunomodulatory polypeptide selected from CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/ TR6, ILT3, ILT4 and HVEM.

Aspect 48. The nucleic acid of any one of aspects 37-47, wherein the proteolytically cleavable linker or ribosome skipping signal comprises an amino acid sequence selected from

a) LEVLFQGP (SEQ ID NO:88);

b) ENLYTQS (SEQ ID NO:90);

c) furin cleavage site;

d) LVPR (SEQ ID NO:89);

e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:91);

f) GSGERGGSLTCGDVEENPGP (SEQ ID NO:92);

g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:93); And

h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:94).

Aspect 49. The nucleic acid of aspect 31, wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus:

a) a first leader peptide;

b) an epitope;

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c) the first MHC polypeptide;

d) an immunomodulatory polypeptide;

f) a proteolytically cleavable linker or ribosome skipping signal;

f) a second leader peptide;

g) a second MHC polypeptide; and

h) an immunoglobulin (Ig) Fc polypeptide.

Aspect 50 The nucleic acid of Aspect 49 wherein the first leader peptide and the second leader peptide are the b2-M leader peptide.

Aspect 51. The nucleic acid of any one of aspects 37-50, wherein the nucleotide sequence is operably linked to a transcription control element.

Aspect 52 The nucleic acid of aspect 51 wherein the transcriptional control element is a promoter that is functional in a eukaryotic cell.

Aspect 53. The nucleic acid of any one of aspects 37-52, wherein the first MHC polypeptide or linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to form a first Cys residue and the second MHC polypeptide comprises an amino acid substitution to form a second Cys residue, and wherein the first and the second Cys residues provide a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

Aspect 54. A recombinant expression vector containing a nucleic acid according to any one of aspects 37-52.

Aspect 55 The recombinant expression vector of aspect 54, wherein the vector is a viral vector or a non-viral vector.

Aspect 56. A host cell genetically modified with a recombinant expression vector according to Aspect 48-55.

Aspect 57. The host cell of aspect 56, wherein the host cell is in vitro.

Aspect 58. The host cell of aspect 57, wherein the host cell is genetically modified such that the cell does not produce endogenous MHC β2-microglobulin polypeptide.

Aspect 59. A composition comprising:

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain, wherein the immunomodulatory domain is a variant of an IL2 polypeptide according to any one of aspects 1-7; and

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect 60: Composition comprising:

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) an immunomodulatory domain, wherein the immunomodulatory domain is a variant of an IL2 polypeptide according to any one of aspects 1-7;

ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide.

Aspect 61 The composition of aspect 59 or aspect 60, wherein the first and/or second nucleic acid is present in the recombinant expression vector.

Aspect 62. A host cell genetically modified with a composition of any of Aspects 5961.

Aspect 63 A method for preparing a multimeric polypeptide according to any one of aspects 8-36, the method comprising:

a) culturing the host cell according to any one of aspects 56-58 and 62 in vitro in a culture medium under conditions under which the host cell synthesizes the multimeric polypeptide; and

b) isolating the multimeric polypeptide from the host cell and/or from the culture medium.

Aspect 64. The method of Aspect 63, wherein the second polypeptide comprises an affinity tag, and wherein said isolation comprises contacting the multimeric polypeptide produced by the cell with a binding partner for the affinity tag, wherein the binding partner is immobilized, thereby

- 120 042111 immobilizing the multimeric polypeptide.

Aspect 65 The method of aspect 64 comprising eluting the immobilized multimeric polypeptide.

Aspect 66. A method for selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any one of aspects 8-36, wherein said contacting selectively activates the epitope-specific T cell.

Aspect 67 The method of aspect 66 wherein said contacting occurs in vitro.

Aspect 68 The method of aspect 66 wherein said contacting occurs in vivo.

Aspect 69 The method of aspect 66 wherein the epitope is a cancer associated epitope and wherein said administration selectively increases the activity of a T cell specific for the cancer associated epitope.

Aspect 70. A method of treating cancer in a subject, comprising administering to the subject an effective amount of:

a) a multimeric polypeptide according to any one of aspects 8-36; or

b) one or more recombinant expression vectors containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 8-36; or

c) one or more mRNAs comprising nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 8-36, wherein the epitope is a cancer associated epitope and wherein said administration is effective to selectively activate an epitope-specific cancer T cell in an individual.

Aspect 71. The method of aspect 70 wherein said administration is subcutaneous.

Aspect 72. The method of aspect 70 wherein said administration is intravenous.

Aspect 73. The method of aspect 70 wherein said administration is intratumorally.

Aspect 74. The method of aspect 70 wherein said administration is systemic.

Aspect 75. The method of aspect 70 wherein said administration is distal to the site of treatment.

Aspect 76. The method of aspect 70 wherein said administration is topically.

Aspect 77. The method of aspect 70 wherein said administration is at or near the treatment site.

Aspect 78. A composition comprising:

a) a multimeric polypeptide according to any one of aspects 8-36; and

b) a pharmaceutically acceptable excipient.

Aspect 79: Composition comprising:

a) a nucleic acid according to any one of aspects 37-53 or a recombinant expression vector according to aspect 54 or 55; and

b) a pharmaceutically acceptable excipient.

Aspect 80. A multimeric polypeptide comprising:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a microglobulin β2 (v2M) polypeptide containing the amino acid sequence depicted in FIG. 34A; and

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) an IL-2 polypeptide variant according to any one of aspects 1-7;

ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S (N77A, L14A, L15A, L14F, L15E, and P111S, respectively, based on the amino acid numbering shown in FIG. 33A).

Aspect 81 The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide contains an N297A substitution (N77A based on the amino acid numbering shown in Figure 33A).

Aspect 82 The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises an L234A substitution and an L235A substitution (L14A and L15A based on the amino acid numbering shown in Figure 33A).

Aspect 83 The multimeric polypeptide of aspect 80 wherein the IgG1 Fc polypeptide comprises an L234F substitution and an L235E substitution (L14F and L15E based on the amino acid numbering shown in Figure 33A).

Aspect 84 The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide comprises an L234F substitution, an L235EHP331S substitution (L14F, L15E and PAP substitutions based on the amino acid numbering shown in Figure 33A).

Aspect 85 A multimeric polypeptide according to any one of aspects 80-84, wherein the second polypeptide contains two copies of a variant IL-2 polypeptide.

Aspect 86 The multimeric polypeptide of any one of aspects 80-85, wherein the first polypeptide comprises a peptide linker between the epitope and the b2M polypeptide.

- 121 042111

Aspect 87 The multimeric polypeptide of any one of aspects 80-86, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and

c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide.

Aspect 88 A multimeric polypeptide according to Aspect 86 or Aspect 87 wherein the peptide linker is selected from (GGGGS)3, (GGGGS) ₄ and AAAGG.

Aspect 89. A multimeric polypeptide comprising:

a) a first polypeptide containing, in order from N-terminus to C-terminus: i) an epitope;

ii) a β2-microglobulin polypeptide comprising the amino acid sequence shown in FIG. 34A; and

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) an IL-2 polypeptide variant containing the amino acid sequence depicted in FIG. 34V;

ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S (N77A, L14A, L15A, L14F, L15E, and P111S, respectively, based on the amino acid numbering shown in FIG. 33A).

Aspect 90 The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide contains an N297A substitution (N77A based on the amino acid numbering shown in Figure 33A).

Aspect 91 The multimeric polypeptide of aspect 89 wherein the IgG1 Fc polypeptide comprises an L234A substitution and an L235A substitution (L14A and L15A based on the amino acid numbering shown in Figure 33A).

Aspect 92 The multimeric polypeptide of aspect 89 wherein the IgG1 Fc polypeptide comprises an L234F substitution and an L235E substitution (L14F and L15E based on the amino acid numbering shown in Figure 33A).

Aspect 93 The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide comprises an L234F substitution, an L235E substitution, and a P331S substitution (L14F, L15E, and P111S based on the amino acid numbering shown in Figure 33A).

Aspect 94 The multimeric polypeptide of any one of aspects 89-93, wherein the second polypeptide contains two copies of a variant IL-2 polypeptide.

Aspect 95 The multimeric polypeptide of any one of aspects 89-94, wherein the first polypeptide comprises a peptide linker between the epitope and the b2M polypeptide.

Aspect 96 The multimeric polypeptide of any one of aspects 89-95, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and

c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide.

Aspect 97 A multimeric polypeptide according to Aspect 95 or Aspect 96 wherein the peptide linker is selected from (GGGGS)3, (GGGGS)4 and AAAGG.

Aspect 98. A multimeric polypeptide comprising:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 77);

ii) a β2-microglobulin polypeptide comprising the amino acid sequence shown in FIG. 34A; and

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) an IL-2 polypeptide variant containing the amino acid sequence depicted in FIG. 34V;

ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence depicted in FIG. 34C; and iii) an IgG1 Fc polypeptide comprising the amino acid sequence depicted in FIG. 33A, 33B, 33C or 33D.

Aspect 99 The multimeric polypeptide of Aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence shown in FIG. 33B.

Aspect 100 The multimeric polypeptide of Aspect 98 wherein the IgG1 Fc polypeptide comprises the amino acid sequence shown in FIG. 33C.

Aspect 101 The multimeric polypeptide of Aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence shown in FIG. 33d.

Aspect 102 The multimeric polypeptide of any one of aspects 98-101, wherein the second polypeptide contains two copies of a variant IL-2 polypeptide.

Aspect 103 The multimeric polypeptide of any one of aspects 98-102, wherein the first polypeptide comprises a peptide linker between the epitope and the b2M polypeptide.

- 122 042111

Aspect 104 The multimeric polypeptide of any one of aspects 98-103, wherein the second polypeptide comprises a peptide linker between one or more of:

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) an IL-2 polypeptide variant and an MHC heavy chain polypeptide; and

c) between an MHC heavy chain polypeptide and an IgG1 Fc polypeptide.

Aspect 105 A multimeric polypeptide according to aspect 103 or aspect 104, wherein the peptide linker is selected from (GGGGS) ₃ , (GGGGS) ₄ and AAAGG. Aspect 106 A multimeric polypeptide comprising:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 22.

Aspect 107 A multimeric polypeptide comprising:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 25.

Aspect 108 A multimeric polypeptide comprising:

a) a first polypeptide containing the amino acid sequence depicted in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 28.

Aspect 109. A pharmaceutical composition comprising:

a) a multimeric polypeptide according to any one of aspects 80-108; and

b) a pharmaceutically acceptable excipient.

Aspect 110. One or more nucleic acids comprising nucleotide sequences encoding the first and/or second polypeptide of a multimeric polypeptide, according to any one of aspects 80-108.

Aspect 111 One or more of the nucleic acids of aspect 110, wherein the nucleic acids are present in recombinant expression vectors.

Aspect 112. A method for selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any one of aspects 80-108, wherein said contacting selectively activates the epitope-specific T cell.

Aspect 113. The method of aspect 112 wherein said contacting occurs in vitro.

Aspect 114. The method of aspect 112 wherein said contacting occurs in vivo.

Aspect 115. A method comprising administering to an individual an effective amount of:

a) a multimeric polypeptide according to any one of aspects 80-108; or

b) one or more recombinant expression vectors containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 80-108; or

c) one or more mRNAs comprising nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 80-108, wherein said administration induces a T cell response to an epitope in an individual.

Aspect 116. The method of aspect 115 wherein said administration is subcutaneous.

Aspect 117. The method of aspect 115 wherein said administration is intravenous.

Aspect 118. The method of aspect 115 wherein said administration is systemic.

Aspect 119 The method of aspect 115 wherein said administration is intramuscularly.

Aspect 120. The method of aspect 115, wherein said administration is distal to the site of treatment.

Aspect 121. The method of aspect 115 wherein said administration is topically.

Aspect 122. The method of aspect 115, wherein said administration is at or near the treatment site.

Aspect A. A method for modulating an immune response in an individual in need thereof, the method comprising administering to the individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration modulates an immune response in an individual.

Aspect B. A method of treatment comprising administering to an individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats the subject.

Aspect C. A method of treating cancer in a subject, comprising administering to the subject a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats cancer in an individual.

Aspect D. A method of treatment comprising administering to an individual a multimeric polypeptide, wherein the individual is being treated with an immune checkpoint inhibitor, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first major histocompatibility complex (MCHC) polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are located:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide; and wherein said administration treats the subject.

Aspect E. The method of any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect F. The method of any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) an immunomodulatory domain;

iii) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide.

Aspect G. The method of any of Aspects A-D, wherein the multimeric polypeptide comprises:

- 124 042111

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) an immunomodulatory domain.

Aspect H. The method of any of Aspects A to D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and ii) an immunomodulatory domain.

Aspect I. The method of any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope; and ii) a first MHC polypeptide; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) an immunomodulatory domain; and ii) a second MHC polypeptide.

Aspect J. The method of any of Aspects A-D, wherein the multimeric polypeptide comprises:

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and iii) an immunomodulatory domain; And

b) a second polypeptide containing, in order from N-terminus to C-terminus:

i) a second MHC polypeptide.

Aspect K The method of any one of aspects A-D, wherein the non-Ig backbone of the multimeric polypeptide is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

Aspect L. The method of any one of A-K, wherein the first MHC polypeptide of the multimeric polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide.

Aspect M. The method of Aspect L, wherein the β2-microglobulin polypeptide of the multimeric polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 6.

Aspect N. The method of aspect L, wherein the MHC class I heavy chain polypeptide of the multimeric polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect O. The method of Aspect 11 wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 5A-5C.

Aspect P. The method of any one of A-K, wherein the first MHC polypeptide of the multimeric polypeptide is an MHC class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

Aspect Q The method of any one of A-P, wherein the epitope is a T cell epitope.

Aspect R. The method of any one of A-J, wherein the multimeric polypeptide of the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgA Fc Fc polypeptide, or an IgM Fc polypeptide.

Aspect S. The method of Aspect R, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence shown in FIG. 4A-4C.

Aspect T. The method of any one of A-S, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are non-covalently linked.

Aspect U The method of any one of A-S, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are covalently linked.

Aspect V. The method of Aspect U wherein the covalent bond is via a disulfide bond.

Aspect W. The method of Aspect V, wherein the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution to obtain

- 125 042111 of the first Cys residue and the second MHC polypeptide of the multimeric polypeptide contains an amino acid substitution to obtain a second Cys residue and where the disulfide bond is between the first and second Cys residues.

Aspect X. The method of any one of A-K, wherein the multimeric polypeptide comprises a linker between an epitope and a first MHC polypeptide, between an immunomodulatory polypeptide and an MHC polypeptide, or between an MHC polypeptide and an Ig Fc.

Aspect Y. The method of any one of A-K, wherein the immunomodulatory polypeptide of the multimeric polypeptide is selected from a 4-1BBL polypeptide, a B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

Aspect Z The method of any one of aspects A-Y, wherein the multimeric polypeptide comprises 2 or more immunomodulatory polypeptides.

Aspect AA. The method according to aspect Z, wherein 2 or more immunomodulatory polypeptides are in tandem.

Aspect VV. The method according to any one of aspects A-Z and AA, wherein the immunomodulatory polypeptide is selected from a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, an IL-2 polypeptide, a B7-1 polypeptide; B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide and PD-L2 polypeptide.

Aspect SS. The method according to any one of aspects A-Z and AA-BB, wherein the immunomodulatory polypeptide is an immunomodulatory polypeptide variant having one or more amino acid substitutions relative to the natural form of the immunomodulatory polypeptide, and wherein the immunomodulatory polypeptide variant exhibits reduced binding affinity for the comodulatory polypeptide to which the natural form binds immunomodulatory polypeptide.

Aspect D.D. The method according to any one of aspects A-Z and AA-BB, wherein the immunomodulatory polypeptide is an IL-2 polypeptide variant comprising an amino acid sequence having at least 85% amino acid sequence identity as specified in SEQ ID NO: 1, wherein the IL-2 polypeptide variant has one or more amino acid substitutions relative to the sequence shown in SEQ ID NO: 1, and where the IL-2 polypeptide variant exhibits reduced binding affinity for the IL-2 receptor (IL2R) containing alpha, beta and gamma polypeptides having amino acid sequences, shown in FIG. 3A-3C compared to the binding affinity of the IL-2 amino acid sequence indicated in one of SEQ ID NO: 1 for IL2R.

Aspect EE. The method of aspect DD, wherein the variant IL-2 polypeptide comprises replacing one or more of E15, H16, D20, F42, Y45, and Q126.

Aspect F.F. The method according to aspect EE, wherein the variant IL-2 polypeptide comprises:

a) F42 and D20 replacements;

b) P42 and H16 substitutions;

c) F42, D20 and Y45 replacements; or

d) F42, H16 and Q126 replacements.

GG aspect. The method according to any one of aspects A-Z and AA-FF, wherein the multimeric polypeptide comprises an Ig Fc polypeptide containing one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S.

Aspect HH. The method according to aspect GG, wherein the Ig Fc polypeptide comprises:

a) N297A replacement;

b) L234A replacement and L235A replacement;

c) L234F replacement and L235E replacement; or

d) L234F replacement, L235E replacement and P331S replacement.

Aspect II. The method according to any one of A-Z and AA-HH, wherein the epitope of the multimeric polypeptide comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 77).

JJ aspect. The method according to any one of aspects A-Z and AA-HH, wherein the β2-microglobulin polypeptide of the multimeric polypeptide comprises the amino acid sequence depicted in FIG. 34A.

Aspect KK. The method of any one of A-Z and AA-JJ, wherein the major histocompatibility complex (MHC) heavy chain polypeptide of the multimeric polypeptide comprises the amino acid sequence shown in FIG. 34C.

Aspect L.L. The method according to any one of aspects A-Z and AA-QC, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor.

Aspect MM. The method of aspect LL, wherein the antibody is a monoclonal antibody.

Aspect N.N. A method according to aspect KK or aspect LL, wherein the antibody comprises at least one humanized light chain and/or heavy chain framework region.

Aspect of OO. The method according to aspect LL, wherein the antibody comprises an Fc polypeptide, and wherein the Ig Fc polypeptide

- 126 042111 is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

PP aspect. The method according to aspect LL, wherein the antibody is an Fv fragment, a nanobody, or a Fab fragment.

QQ aspect. The method according to any one of the aspects of LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

Aspect R.R. The method according to any one of the aspects of LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for PD1.

Aspect SS. The method according to aspect RR, wherein the antibody is pembrolizumab, nivolumab, pidilizumab, or BMS-39886.

Aspect TT. The method according to any one of the aspects of LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1.

UU aspect. The method according to aspect TT, wherein the antibody is durvalumab, atezolizumab, KN035, or avelumab.

Aspect V.V. The method according to any one of the aspects of LL-PP, wherein the immune checkpoint inhibitor is an antibody specific for CTLA4.

W.W. aspect. The method of aspect VV, wherein the antibody is ipilimumab or tremelimumab.

Aspect XX. The method of any one of aspects A-Z and AA-WW, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by the same route of administration.

Aspect YY. The method according to any one of aspects A-Z and AA-WW, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by various routes of administration.

ZZ aspect. The method of any one of aspects A-Z and AA-YY, wherein the multimeric polypeptide is administered by an administration route selected from subcutaneous, intravenous, peritumoral, and intramuscular.

AAA aspect. The method of any one of aspects A-Z and AA-YY, wherein the immune checkpoint inhibitor is administered by an administration route selected from subcutaneous, intravenous, peritumoral, and intramuscular.

Aspect VVV. The method according to any one of aspects A-Z, AA-ZZ and AAA, wherein the subject is a human.

Examples

The following examples are provided to provide those skilled in the art with a full disclosure and description of how to make and use the present invention, however, they are not intended to limit the scope of what the authors consider their invention to be, nor that the following experiments represent all or the only experiments performed. Care has been taken to maintain accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts mean parts by weight, molecular weight means average molecular weight, temperature is expressed in degrees Celsius, and pressure represents atmospheric or near atmospheric pressure. Standard abbreviations may be used, eg b.p., base pair(s); etc. n., thousand (and) pairs of nucleotides; pl, picoliter(s); s or sec, second(s); min, minute(s); h or hour, hour(s); ak, amino acid(s); thousand (and) pairs of nucleotides; b.p., pair(s) of bases; nt, nucleotide(s); kiloDalton(s), kDa; i / m, intramuscular (o); i/b, intraperitoneal (o); s / c, subcutaneous (o), etc.

Example 1 Production of IL-2/synTac.

The production of IL-2/synTac by transiently transfected mammalian cells was analyzed. As shown in FIG. 7A, production levels (in mg/L of culture medium) of two different IL2/synTac 6-7 days after transient transfection of cells were over 90 mg/L.

IL-2/synTac produced by mammalian cells was purified and subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The results are shown in FIG. 7B. Dimensions are in kDa.

An IL-2/synTac was prepared in which the IL-2 polypeptide was in the light chain (i.e., in a polypeptide containing an MHC class I light chain; e.g., B2M) or in the heavy chain (i.e., in a polypeptide containing heavy chain MHC class I). Expression levels and stability of IL-2/synTac were analyzed.

synTac was produced in mammalian cells. As shown in FIG. 8A, the level of production of IL2/synTac containing IL-2 in the heavy chain was approximately 25 times higher than that of IL-2/synTac containing IL-2 in the light chain.

IL-2/synTac produced by mammalian cells was subjected to polyacrylamide gel electrophoresis under reducing and non-reducing conditions; and gels stained with Coomassie

- 127 042111 blue. As shown in FIG. 8B, IL-2/synTac containing IL-2 in the heavy chain was more stable than IL2/synTac containing IL-2 in the light chain. Dimensions are in kDa.

Expression levels of IL-2/synTac containing the IL-2 variant were assessed. In FIG. 9 shows the expression level of IL-2/synTac in which IL-2 is wild-type (wt) or contains various combinations of F42A, D20K, Q126A, E15A, Y45A and H16A. Expression levels are expressed as percentage change relative to synTac expression levels with wild-type IL-2.

The effect of IL-2 copy number in IL-2/synTac on expression levels was evaluated. IL-2/synTac contained one copy (1X), two copies (2X) or three copies (3X) in synTac. Various IL-2/synTacs were produced in mammalian cells and expression levels were analyzed. The data is shown in Fig. 10. IL-2/synTac with one or two copies of IL-2 showed similar levels of expression, while IL-2/synTac with three copies of IL-2 showed lower levels of expression. Expression levels are expressed as a fold change relative to the level of expression of IL-2/synTac with one copy of IL-2.

Example 2 Activity of IL-2/synTac in vitro.

To achieve maximum targeting specificity through the T cell receptor, the affinity of the co-stimulatory polypeptide for its ligand must be lower than the affinity of MHC for the TCR. The affinity of the peptide/MHC to TCR can be about 10 μm.

IL-2/synTac was created containing two copies of the IL-2 variant containing F42A and H16A substitutions. Costimulatory signaling induced by IL-2/synTac has been tested on antigen-specific CD8+ T cells and non-specific CD8+ T cells. Antigen-specific CD8+ T cells and non-specific CD8+ T cells have been associated with various concentrations of IL-2/synTac.

As shown in FIG. 11, IL-2/synTac induced costimulatory signaling in antigen-specific CD8+ T cells at a significantly lower concentration than in non-specific CD8+ T cells.

The binding selectivity of IL-2/synTac was tested. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. CD8+ T cells were incubated with several IL-2/synTacs at various concentrations and allowed to bind for 20 minutes. Several IL-2/synTacs contained IgG2a Fc. Binding of IL-2/synTac to CD8+ T cells was detected using a phycoerythrin (PE) labeled anti-IgG2a antibody. PE fluorescence was determined using flow cytometry to determine the percentage of cells associated with IL-2/synTac.

As shown in FIG. 12, IL-2/synTac binds in an antigen-specific manner to LCMV CD8+ T cells, but shows no significant binding to OT1 CD8 ⁺ T cells. Thus, IL2/synTac selectively binds to CD8+ T cells specific for an epitope present in IL2/synTac.

It was determined whether IL-2/synTac is able to selectively activate target T cells. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. The IL-2/synTacs used included either a single F42A amino acid substitution or F42A and H16A substitutions. CD8+ T cells were stimulated with IL-2/synTac at various concentrations for 20 min. Cells were then stained with PE-labeled anti-phosphoSTAT5 antibody. PE fluorescence was determined using flow cytometry to determine the percentage of cells that are phospho-STAT5 positive, where phospho-STAT5 is an activation marker.

As shown in FIG. 13, IL-2/synTac induced CD8+ stimulation (as indicated by % phosphoSTAT5 positive cells) in antigen-specific (LCMV) CD8 ⁺ T cells at significantly lower concentrations than in non-specific (BL6) CD8 ⁺ T cells.

The specific activity of various IL-2/synTacs was analyzed. IL-2/synTac containing one copy of IL-2, two copies of IL-2, or three copies of IL-2, where IL-2 contained various combinations of F42A, D20K, Q126A, E15A, H16A, and Y45A substitutions were tested at different concentrations. to assess stimulation of antigen-specific (LCMV) or non-specific (BL6) CD8+ cells. The percentage of 5 (pSTAT5)-positive phosphosignal transducer and transcription activator was determined. The data is shown in Fig. 14A-14F.

Example 3 Activity of IL-2/synTac in vivo.

IL-2/synTac activity was tested in vivo. An in vivo fold change was tested in antigen-specific CD8 ⁺ T cells following administration of phosphate buffered saline (PBS), recombinant IL-2 (rIL-2) or IL-2/synTac of the present invention. The data is shown in FIG. 15, left panel. The data show that IL-2/synTac is 10 times more effective than rIL-2.

The specificity of IL-2/synTac was tested in vivo. Antigen-specific and non-antigen-specific responses were evaluated after administration of PBS, rIL-2 or IL-2/synTac. Data are expressed as the percentage of lymph node cells that were antigen-specific or antigen-non-specific after administration of PBS, rIL-2, or IL-2/synTac. As shown in FIG. 15, right panel, IL-2/synTac induced an antigen-specific response (expressed as % maximum dilution of carboxyfluorescein succinimidyl ester (CFSE) as an index of T cell proliferation). In contrast, the response induced by rIL-2 was not antigen specific.

A dose-response analysis was performed. IL-2/synTac (F42A, H16A) was administered intraperitoneally at concentrations of 4 mg/kg, 8 mg/kg and 16 mg/kg. The results are shown in FIG. 16A. As shown in FIG. 16A, IL-128 042111

2/synTac given at 4 mg/kg or 8 mg/kg gave similar results; IL-2/synTac, administered at a dose of 16 mg/kg, produced the strongest immunostimulatory activity.

The effect of the route of administration of IL-2/synTac was tested. IL-2/synTac (F42A, H16A) was administered at a dose of mg/kg either subcutaneously (SubQ) or intraperitoneally (IP). As shown in FIG. 16B, subcutaneous administration resulted in stronger immunostimulatory activity than intraperitoneal administration.

The effect of the number of copies of IL-2 on efficiency has been established. IL-2/synTac containing one copy of IL-2 (F42A, H16A) or two copies of IL-2 (F42A, H16A) were administered to mice with tumors bearing the HPV E7 epitope. The epitope included in these IL-2/synTacs was the HPV E7 epitope. As shown in FIG. 17A and 17B, IL-2/synTac containing two copies of IL-2 (F42A, H16A) was more effective in reducing tumor size than IL-2/synTac containing only one copy of IL-2 (F42A, H16A) .

Example 4 PK/PD and IL-2/synTac stability studies.

A pharmacokinetic (PK) analysis of IL-2/synTac was performed. IL-2/synTac (F42A, D20K, H16A) was administered intraperitoneally (IP) at a dose of 10 mg/kg. Serum samples were obtained at various time points after administration and the level of IL-2/synTac was measured in the serum samples. As shown in FIG. 18, the serum half-life of IL-2/synTac was approximately 4 hours.

IL-2/synTac was administered IP to C57BL/6 mice at a dose of 10 mg/kg and serum was collected two hours after injection. IL-2/synTac included a His6 tag. 100 ng of the injected protein, or the equivalent of 40 μl of serum, was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and examined with anti-(His)6 antibody or anti-β-2M antibody. The results shown in FIG. 19 show that IL-2/synTac remains stable and intact for at least 2 hours in vivo.

IL-2/synTac was stored at 4°C or 37°C for 5 days. Analyzed 0.5 mg of each sample (at 10 mg/ml) by size exclusion chromatography. As shown in FIG. 20, IL-2/synTac is stable and intact for at least 5 days at 4°C or 37°C.

Example 5 Effect of IL-2/synTac and anti-PD1 antibody on tumor volume.

As shown in FIG. 35, administration of IL-2/synTac and anti-PD1 antibody to tumor mice reduced tumor volume.

Example 6 Preparation and characterization of synTac polypeptides with variant 4-1BBL SynTac polypeptides were synthesized and characterized. The following synTac polypeptides have been tested for activity against ovalbumin-specific (OVA) T cells:

1) Syn83/51. The Syn83/51 light chain contains: a) an OVA T-cell epitope; b) amino acids 50-254 of the wild type 4-1BBL polypeptide; and c) v2M; and the heavy chain of Syn83/51 contains: a) the heavy chain of MHC; and b) Fc Ig.

2) Syn239/345. The light chain Syn239/345 contains: a) an epitope of OVA T cells; b) trimer from amino acids 80-254 4-1BBL wild type; and c) v2M; and the heavy chain of Syn239/345 contains: a) the heavy chain of MHC; and b) FcIgG2a.

3) Syn341/348. The Syn341/348 light chain contains: a) an OVA T-cell epitope; b) wild type 4-1BBL trimer; and c) v2M; and the heavy chain of Syn239/345 contains: a) the heavy chain of MHC; and b) Fc IgG2a. In Syn341/348, the first unit of the 4-1BBL trimer contains amino acids 50-254 of the wild-type 4-1BBL; the second and third units of the 4-1BBL trimer contain amino acids 80 to 254 of wild-type 4-1BBL.

4) Syn341/349. The Syn341/349 light chain contains: an OVA T-cell epitope; b) a trimer of amino acids 80-254 4-1BBL containing a K127A substitution in each trimer unit, with a GlySerSerSerSer linker between the first and second units and between the second and third units of the trimer; and c) v2M; and the heavy chain of Syn239/345 contains: a) the heavy chain of MHC; and b) Fc IgG2a.

The resulting synTac heterodimers were cultured in vitro with ovalbumin-specific T cells for 3 days or 5 days at concentrations of 0; 1; 3.17; 10.01; 31.65 and 100 nM synTac. Control included: a) medium only; b) phorbol-12-myristate-13-acetate (PMA) and ionophore A23187; and c) an anti-CD3 antibody and an anti-CD28 antibody.

After 3 days and after 5 days, the concentration of IFN-γ, IL-2, IL-6, TNF, IL-10, IL-17A and IL-4 was determined in the culture medium. In addition, the viability of OVA-specific T cells and the proliferation of OVA-specific T cells were determined.

The data is shown in Fig. 38-46.

As shown in FIG. 38-46, Syn 341/349 induces the production of IL-2 (cellular fitness cytokine); induces the production of cytotoxic cytokines TNFa and IFN-γ; and also induces proliferation and increases the viability of epitope-specific T cells.

Example 7 SynTac production in CHO cells.

SynTacs containing 4-1BBL wild-type (wt) or containing 4-1BBL with amino acid substitutions as shown in FIG. 47 were transiently expressed in CHO cells. The amount of synTac produced was determined. The quantities obtained are shown in Fig. 47.

Example 8 Effect of synTac 4-1BBL in vivo.

Injected synTac containing antigenic peptide E7 human papillomavirus (HPV) and variant

- 129 042111

K127A 4-1BBL of the present invention (referred to as CUE: 4-1BBL (K127A) in Fig. 48), at a dose of mg/kg by intraperitoneal (IP) injection in mice with lateral-grafted HPV ⁺ TC-1 lung carcinoma. As a control, mice with the same tumor were injected with phosphate-buffered saline (PBS).

As shown in FIG. 48, tumor volume in mice treated with CUE:4-1BBL (K127A) decreased compared to mice treated with PBS.

Example 9 In vivo effects of co-administration of synTac 4-1BBL and an immune checkpoint inhibitor.

As shown in FIG. 49, co-administration of synTac 4-1BBL of the present invention and an anti-PD1 antibody reduced tumor volume in a mouse tumor model and increased the percentage of tumor infiltrating lymphocytes (TILs) that were granzyme B ⁺ .

While the present invention has been described with reference to specific embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of this invention. In addition, numerous modifications may be made to adapt a particular situation, material, chemical composition, process, process step or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims (22)

CLAIM 1. A method for modulating an immune response or treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a composition containing a multimeric polypeptide, where the composition of the multimeric polypeptide contains a heterodimeric polypeptide, where the heterodimeric polypeptide contains: a) a first polypeptide containing: i) a peptide epitope containing the human papillomavirus E7 epitope; and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, and wherein said first and/or said second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide, wherein at least one of the one or more immunomodulatory polypeptides is an IL-2 polypeptide variant that exhibits reduced binding affinity for the IL-2 receptor (IL2R) containing α, β and γ polypeptides having the amino acid sequences shown in FIG. 3A-3C, compared to the binding affinity of IL-2, the amino acid sequence is shown in SEQ ID NO: 1, with IL2R, where one or more linkers can be placed between one or more constituents of said first and second polypeptides, and where an inhibitor of the immune control dots and the composition containing the multimeric polypeptide may be administered simultaneously or at different points in time. 2. A method for modulating an immune response or treating cancer in a patient who has been treated with a therapeutically effective amount of an immune checkpoint inhibitor composition, wherein the method comprises administering to the patient a therapeutically effective amount of a composition comprising a multimeric polypeptide, wherein the multimeric polypeptide composition comprises a heterodimeric polypeptide, wherein the heterodimeric polypeptide comprises : a) a first polypeptide containing: i) a peptide epitope containing the human papillomavirus E7 epitope; and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, and wherein said first and/or said second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide, wherein at least one of the one or more immunomodulatory polypeptides is an IL2 polypeptide variant that exhibits reduced binding affinity to IL-2 receptor - 130 042111 (IL2R) containing α, β and γ polypeptides having the amino acid sequences shown in FIG. 3A-3C, compared to the binding affinity of IL-2, the amino acid sequence is shown in SEQ ID NO: 1, with IL2R, where one or more linkers can be placed between one or more constituents of said first and second polypeptides, and where an inhibitor of the immune control dots and the composition containing the multimeric polypeptide may be administered simultaneously or at different points in time. 3. A method for modulating an immune response or treating cancer in a patient who has been treated with a therapeutically effective amount of a composition comprising a multimeric polypeptide, wherein the method comprises administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor, wherein the multimeric polypeptide composition comprises a heterodimeric polypeptide, wherein the heterodimeric polypeptide comprises: a) a first polypeptide containing: i) a peptide epitope containing the human papillomavirus E7 epitope; and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; wherein the multimeric polypeptide comprises one or more immunomodulatory polypeptides, and wherein said first and/or said second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide, wherein at least one of the one or more immunomodulatory polypeptides is an IL2 polypeptide variant that exhibits reduced binding affinity to an IL-2 receptor (IL2R) containing α, β and γ polypeptides having the amino acid sequences shown in FIG. 3A-3C, compared to IL-2 binding affinity, the amino acid sequence is shown in SEQ ID NO: 1, with IL2R, where one or more linkers can be placed between one or more constituents of said first and second polypeptides, and where an inhibitor of the immune control dots and the composition containing the multimeric polypeptide may be administered simultaneously or at different points in time. 4. The method according to any one of claims 1 to 3, wherein said second polypeptide comprises one or two immunomodulatory polypeptides, and wherein each of the immunomodulatory polypeptides is selected from the group consisting of an IL-2 polypeptide variant containing an H16A substitution relative to the amino acid sequence shown in SEQ ID NO: 1, and an IL-2 polypeptide variant containing H16A and F42A substitutions relative to the amino acid sequence shown in SEQ ID NO: 1. 5. The method according to any one of claims 1-4, wherein the peptide epitope comprises an amino acid sequence selected from the group consisting of HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO: 75), HPV16E7/86-93 (TLGIVCPI; SEQ ID NO: 76), HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO: 77), and HPV16E7/11-19 (YMLDLQPET; SEQ ID NO: 78). 6. The method according to any one of claims 1 to 5, wherein the multimeric polypeptide comprises an Ig Fc polypeptide, wherein the Ig Fc polypeptide comprises the amino acid substitutions L234A and L235A. 7. A method according to any one of claims 1-6, wherein the β2-microglobulin polypeptide has at least 95% sequence identity with amino acids 21-119 of any of the amino acid sequences shown in FIG. 6. 8. The method of any one of claims 1-7, wherein the MHC class I heavy chain polypeptide is an HLA-A heavy chain. 9. The method according to any one of claims 1 to 8, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 59. 10. The method of any one of claims 1-9, wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond, wherein the disulfide bond connects a Cys residue in the b2M polypeptide and a Cys residue in the MHC heavy chain polypeptide. 11. The method of claim 10 wherein the Cys at amino acid residue 12 of the b2M polypeptide is disulfide-linked to the Cys at amino acid residue 236 of the MHC heavy chain polypeptide. 12. The method of claim 10, wherein the first polypeptide chain comprises a linker between the peptide epitope and the b2M polypeptide, and wherein the disulfide bond links the Cys present in the linker to the Cys of the MHC heavy chain polypeptide. 13. The method according to any one of claims 1-3, in which the multimeric polypeptide contains: - 131 042111 a) the first polypeptide containing, in order from N-terminus to C-terminus: i) a peptide epitope, wherein the peptide epitope contains the amino acid sequence YMLDLQPETT (SEQ ID NO: 77) and is 10 amino acids long; and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first polypeptide MHC is a β2-microglobulin polypeptide; b) a second polypeptide containing, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide, wherein the first immunomodulatory polypeptide is an IL-2 polypeptide variant containing an H16A substitution and an F42A substitution relative to the amino acid sequence shown in SEQ ID NO: 1; ii) a second immunomodulatory polypeptide, wherein the second immunomodulatory polypeptide is an IL-2 polypeptide variant containing an H16A substitution and an F42A substitution relative to the amino acid sequence shown in SEQ ID NO: 1; iii) a second MHC polypeptide, wherein the second polypeptide is an MHC class I heavy chain polypeptide; and iv) an immunoglobulin (Ig) Fc polypeptide, and wherein the first polypeptide comprises a linker between an epitope and a β2-microglobulin polypeptide, and wherein the second polypeptide comprises a peptide linker between one or more of: a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide; b) a variant IL-2 polypeptide and an MHC heavy chain polypeptide; And c) an MHC heavy chain polypeptide and an IgG1 Fc polypeptide, wherein the β2-microglobulin polypeptide has at least 95% sequence identity with amino acids 21-119 of any of the amino acid sequences shown in FIG. 6, the MHC class I heavy chain polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 59, and the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond, and where a disulfide bond connects at amino acid residue position 12 of the b2M polypeptide to a Cys residue at amino acid residue position 236 of the MHC heavy chain polypeptide. 14. The method according to claim 13, in which: i) the β2-microglobulin polypeptide contains the amino acid sequence depicted in FIG. 34A; ii) the first and second variant IL-2 polypeptides contain the amino acid sequence depicted in FIG. 34V; iii) the MHC class I heavy chain polypeptide contains the amino acid sequence depicted in FIG. 34C; and iv) the Ig Fc polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence shown in FIG. 33A, 33B, 33C or 33D. 15. The method of claim 13, wherein the amino acid sequence of the b2M polypeptide comprises an R12C substitution and the MHC class I heavy chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 50. 16. The method of claim 15 wherein the peptide linker is selected from (GGGGS) ₃ , (GGGGS) ₄ and AAAGG. 17. The method according to any one of claims 13-16, wherein the multimeric polypeptide comprises: a) the first polypeptide containing, in order from N-terminus to C-terminus: i) an HPV16 E7 epitope containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 77); ii) a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 207); and iii) a b2M polypeptide comprising the amino acid sequence shown in SEQ ID NO: 48; And b) a second polypeptide containing an amino acid sequence selected from the amino acid sequence shown in FIG. 22 of the amino acid sequence shown in FIG. 25 of the amino acid sequence shown in FIG. 28. 18. The method of any one of claims 1 to 17, wherein the composition comprising the multimeric polypeptide comprises a protein comprising said two heterodimeric polypeptides, and wherein said two heterodimeric polypeptides each comprise an immunoglobulin (Ig) Fc polypeptide. 19. The method according to any one of claims 1 to 18, wherein the two heterodimeric polypeptides are disulfide-linked to each other via the Fc polypeptides present in the heterodimers. 20. The method of any one of claims 1-19, wherein the immune checkpoint inhibitor is an antibody that specifically binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR , CSF1R, JAK, PI3Kδ, PI3Ky, TAM, arginases, - 132 042111 CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. 21. The method of any one of claims 1 to 19, wherein the immune checkpoint inhibitor is an antibody specific for PD1. 22. The method of any one of claims 1-19, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1 or CTLA4.