WO2022125694A1

WO2022125694A1 - Fusions of interleukin polypeptides with bispecific antigen binding molecules for modulating immune cell function

Info

Publication number: WO2022125694A1
Application number: PCT/US2021/062458
Authority: WO
Inventors: Yik Andy Yeung; Ivana DJURETIC
Original assignee: Asher Biotherapeutics, Inc.
Priority date: 2020-12-09
Filing date: 2021-12-08
Publication date: 2022-06-16

Abstract

The present disclosure relates to fusion polypeptides comprising a bispecific antigen binding molecule that binds CDS and an activation marker expressed on CD8+ T cells fused to an immunomodulatory peptide, as well as methods, polynucleotides, vectors, host cells, pharmaceutical compositions, and uses related thereto.

Description

FUSIONS OF INTERLEUKIN POLYPEPTIDES WITH BISPECIFIC ANTIGEN BINDING MOLECULES FOR MODULATING IMMUNE CELL FUNCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63/123,388, filed December 9, 2020, which is hereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 182842000640SEQLIST.TXT, date recorded: December 6, 2021, size: 27,676 bytes).

FIELD

[0003] The present disclosure provides fusion polypeptides comprising a bispecific antigen binding molecule that binds CD8 and an activation marker expressed on CD8+ T cells fused to an immunomodulatory peptide. The present disclosure provides methods of modulating immune cell function by contacting the immune cell with fusion polypeptides of the present disclosure. In addition, the disclosure also provides polynucleotides encoding the disclosed fusion molecules, and vectors and host cells comprising such polynucleotides. The present disclosure further provides methods for producing the fusion molecules, pharmaceutical compositions comprising the same, and uses thereof.

BACKGROUND

[0004] lnterleukin-2 (IL-2) is a cytokine that regulates many lymphocyte subsets, including alpha beta CD4+ and CD8 T+ cells, and various innate and innate-like lymphocytes such as NK cells, NK T cells, gamma delta T cells (Tyδ) cells, and innate lymphoid cells (ILC1, ILC2, and ILC3 cells). Binding of IL-2 to its receptor induces the phosphorylation of receptor-associated Janus kinases, JAK3 and JAK1, which promote the phosphorylation of STATS transcription factor (pSTATS) that regulates transcription of many genes in lymphocytes. In addition to STATS, binding of IL-2 to its receptor also activates other signaling pathways such as ERK, PI3K, and Akt kinases. IL-2 signaling in lymphocytes promotes cell survival, proliferation, and increased effector function, including pro-inflammatory cytokine secretion and cytotoxic function, and in some cases, activation-induced cell death (reviewed in Ross & Cantrell, Annu Rev Immunol. 2018 Apr 26;36:411-433).

[0005] IL-2 can signal by binding with an intermediate affinity to a receptor complex consisting of IL-2R and IL-2Rγ subunits (IL-2Rβγ, intermediate affinity receptor), both of which are required and sufficient to trigger downstream signaling in immune cells. In addition, IL-2 binds with high affinity to a receptor complex consisting of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits (IL- 2Rαβγ, high affinity receptor) (Stauber et al, Proc Natl Acad Sci U S A. 2006 Feb 21; 103(8):2788- 93). IL-2Rα expression is restricted to CD4+ Treg cells, activated T lymphocytes, and ILC2 and ILC3 cells, making these subsets the most sensitive to IL-2 signaling. IL-2Rβ and IL-2Rγ subunits are shared with another related cytokine, IL-15, and IL-2Rγ subunit is shared among other common gamma chain cytokines (IL-4, IL-7, IL-9, and IL-21). Most innate and innate-like lymphocytes including NK cells, NK T cells, Ty6 cells, and ILC1, ILC2, and ILC3 cells express high levels of IL-2Rβ (ImmGen consortium; Heng TS et al, Immunological Genome Project Consortium. Nat Immunol. 2008 Oct;9(10):1091-4), which also makes them sensitive to both IL- 2 and IL-15 cytokines.

[0006] In agreement with its potent activities on lymphocytes, systemic administration of high dose of IL-2 resulted in activation of anti-tumor immune responses and efficacy in many preclinical cancer models. Systemically administered high dose IL-2 has also been tested in patients and high dose IL-2 is approved for the treatment of metastatic melanoma and renal cell carcinoma (RCC). Dosing regimen consisted of intravenous injections of 600,000 lU/kg every 8 hr, established based on the in vivo half-life of IL-2 in order to maintain serum levels at concentrations necessary to stimulate high-affinity IL-2 receptors. Overall response rate in RCC was 20% with complete response rate of 9%, while overall response rate in melanoma was 16% with complete response rate of 6% (reviewed in Rosenberg, J Immunol. 2014 Jun

15; 192(12):5451-8). The efficacy of high dose IL-2 in cancer is attributed to its ability to potently expand T cells and NK cells while maintaining their function. However, IL-2 also expands Treg cells and promotes their proper suppressive function (Chinen et al, Nat Immunol. 2016 Nov;17(ll):1322-1333). In fact, due to the sensitivity of Tregs to IL-2, low dose IL-2 therapeutic regimens have been tested in patients with autoimmunity to suppress the pathogenic immune responses (Collison, Nat Rev Rheumatol. 2019 Jan; 15(1):2).

[0007] In addition to its undesired effects on immune-suppressive Treg cells, the benefits of IL- 2 in patients are accompanied by severe toxicity, including fever, chills, malaise, arthralgias, hypotension abnormal liver function, renal failure, and capillary leak syndrome and fluid retention. IL-2 induced-toxicities set the limitation on the number of doses that patients could receive, and IL-2 treatment requires strict patient-eligibility criteria and administration by experienced physicians (Schwartz et al, Oncology (Williston Park). 2002 Nov;16(11 Suppl 13):11- 20). The toxicity of IL-2 involves a complex set of interactions between the immune cells and the vascular endothelium: IL-2-activated cells strongly bind to endothelial cells leading to their lysis, and IL-2 induces pulmonary edema via its interaction with functional IL-2 receptors on endothelial cells (reviewed in Milling et al, Adv Drug Deliv Rev. 2017 May 15; 114: 79-101). Blocking of the IL-2 interaction with IL-2Rα abrogated pulmonary edema in animal models (Krieg et al, Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11906-ll). In addition, the same study showed that blockade of IL-2Rα also led to vigorous activation of IL-2Rβy+ effector immune cells, CD8+ T cells and NK cells, and to a lesser extent, also Tregs, substantially improving both safety and anti-tumor efficacy compared to recombinant IL-2.

[0008] CD8+ T cells have been shown to mediate efficacy of immunotherapeutic agents, including cytokines such as IL-2, in many preclinical cancer models (Caudana et al, Cancer Immunol Res. 2019 Mar;7(3):443-457), and they have also been correlated with response to immunotherapies in patients (Sade-Feldman et al, Cell. 2018 Nov l;175(4):998-1013). CD8+ T cells express CD8, which is a type I transmembrane glycoprotein found on the cell surface as a CD8 alpha (CD8α, CD8a) homodimer and CD8 alpha-CD8 beta (CD8β, CD8b) heterodimer. CD8 dimers interact with the major histocompatibility (MHC) class I molecules on target cells and this interaction keeps the TCR closely engaged with MHC during CD8⁺ T cell activation. The cytoplasmic tail of CD8α contains binding sites for a T cell kinase (Lek) that initiates signal transduction downstream of the TCR during T cell activation, while the role of CD8β is thought to be in increasing the avidity of CD8 binding to MHC class I and influencing specificity of the CD8/MHC/TCR interaction (Bosselut et al, Immunity. 2000 Apr;12(4):409-18). [0009] Intratumoral T cells were recently shown to express activation markers such as PD1 in multiple human cancers (Gros et al, J Clin Invest. 2014 May;124(5):2246-59; Egelston etl al, Nat Commun. 2018 Oct 16;9(1):4297; Thommen et al, Nat Med. 2018 Jul;24(7):994-1004). PD1 is a type I transmembrane protein that contains an extracellular domain, a transmembrane region and a cytoplasmic tail. The cytoplasmic tail contains phosphorylation sites that are part of an immunoreceptor tyrosine-based inhibitory motif (ITIM) that can recruit intracellular phosphatases such as SHP-1 and SHP-2. PD1 negatively regulates TCR signaling by binding to its ligands PD-L1 and PD-L2. The interaction between PD1 and its ligands is blocked by several approved anti-PD1 and anti-PD-Ll antibodies as a treatment for cancer (Ribas & Wolchok, Science. 2018 Mar 23;359(6382):1350-1355).

[0010] High expression of PD1 on intratumoral T cells is associated with specificity for tumor antigens, and the frequency of these PD1+ T cells in tumors was associate with response to anti-PD1 antibodies (Thommen et al, Nat Med. 2018 J u I; 24(7):994-1004) . PD1 is also expressed on peripheral blood CD8+ and CD4+ memory and effector T cells, albeit at a lower level than on tumor antigen-specific intratumoral T cells, and it can also be expressed on T cells residing in healthy tissues. In addition, other cell types such as Tregs, Tyδ, NK T and ILC2 cells can also express PD1.

[0011] In addition to PD1, proteins such as CD137, CD39, TIM3, CD69, CD103, and LAG3 that typically mark antigen/TCR-activated CD8+ T cells were also shown to be enriched on intratumoral CD8+ T cells. Similar to PD1, all four markers were also shown to be expressed on immunosuppressive CD4+ T regulatory cells (Tregs) found in tumors counteracting CD8+ T cells responses and suppressing anti-tumor immunity.

[0012] The goal of this invention was to reduce the toxicity and improve the therapeutic efficacy of cytokines like IL-2 by enhancing their activity on subsets of CD8+ T cells containing tumor antigen-reactive cells. These CD8+ T cells subsets are enriched in tumors and have been associated with efficacy in preclinical cancer models and cancer patients. They can be identified by their higher expression of activation markers such as PD1, CD137, CD39, TIM3, CD69, CD103, and LAG3 (e.g. CD8+PD1+, CD8+CD137+, CD8+CD39+, CD8+TIM3+, CD8+CD69+, CD8+CD103+, CD8+LAG3+ T cells). The bispecific antibodies and fusion molecules of the invention aim to selectively stimulate these activated CD8+ T cells subsets hereby selectively increasing their activity while at the same time reducing their stimulation on other CD8+ T cells that may not contribute to toxic effects more than efficacy and other activated T cells subsets such as Tregs that could negatively impact immune responses against tumors.

[0013] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

[0014] In some aspects, provided herein are fusion proteins comprising a bispecific antigen binding molecule and an immunomodulatory polypeptide. In some embodiments, the bispecific antigen binding molecule comprises a first antigen binding domain that binds to CD8, and a second antigen binding domain that binds to an activation marker expressed on CD8+ T cells. In some embodiments, the bispecific antigen binding molecule is fused to the immunomodulatory polypeptide (e.g., directly or via a linker). In some embodiments, the fusion protein selectively activates CD8+ T cells expressing the activation marker over immune cells expressing only CD8 or only the activation marker. In some embodiments, the CD8+ T cells further express a receptor for the immunomodulatory polypeptide, and the fusion protein activates the immune cells by activation of the receptor via the immunomodulatory polypeptide.

[0015] In some embodiments according to any of the embodiments described herein, the activation marker is selected from the group consisting of PD1, CD137, CD39, CD69, CD103, LAG3, and TIM3. In some embodiments, the immunomodulatory polypeptide is selected from the group consisting of IL-2, IL-7, IL-10, IL-15, IL-18, IL-21, and mutants thereof, wherein the mutants of IL-2, IL-7, IL-10, IL-15, IL-18, or IL-21 are capable of activating signaling via the corresponding receptor. In some embodiments, said immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rα polypeptide comprising the amino acid sequence of SEQ ID NO:2, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL- 2Rα polypeptide. In some embodiments, said immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:3, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL-2Rβ polypeptide. In some embodiments, said mutant immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO:4, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL-2Rγ polypeptide. In some embodiments, said immunomodulatory polypeptide is an IL-2Rβγ agonist polypeptide that binds to and/or activates an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:3; and/or an IL-2Rβγ polypeptide agonist polypeptide that binds to and/or activates an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, said mutant IL-2 polypeptide comprises one or more amino acid substitutions relative to a wild-type IL-2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:1, and wherein the one or more amino acid substitution(s) are at one or more position(s) selected from the group consisting of: Qll, E15, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, N88, V91, 192, T123, Q126, S127, 1129, S130, according to the wild-type IL-2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, said mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:1 with one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E62Q; and F42A, E62Q, and Q126S. In some embodiments, the mutant IL-2 polypeptide comprises a further amino acid substitution relative to SEQ ID NO:1 at position C125. In some embodiments, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:1 with one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO:1): R38E, F42A, and C125A; R38D, F42A , and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A , C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, V91E, and C125A;

F42A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A and Q126S. In some embodiments, the mutant IL-2 polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:7). In some embodiments, said immunomodulatory polypeptide is a mutant IL-21 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-21R polypeptide comprising the amino acid sequence of SEQ ID NO:6, compared to binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO:5 to the IL-21R polypeptide. In some embodiments, said immunomodulatory polypeptide is a mutant IL-21 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rg polypeptide comprising the amino acid sequence of SEQ ID NO:4, compared to binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO:5 to the IL-2Rg polypeptide. In some embodiments, wild type IL-21 comprises the sequence

NO:14). In some embodiments, wild-type IL-21R comprises the sequence

[0016] In some embodiments according to any of the embodiments described herein, the bispecific antigen binding molecule comprises: a first antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

VH1-CH1-hinge-CH2-CH3 [I]; a first antibody light chain polypeptide comprising a structure according to formula [II], from N-terminus to C-terminus:

VL1-CL [ll]; a second antibody heavy chain polypeptide comprising a structure according to formula

[III], from N-terminus to C-terminus:

VH2-CH1-hinge-CH2-CH3 [III]; and a second antibody light chain polypeptide comprising a structure according to formula

[IV], from N-terminus to C-terminus:

VL2-CL [IV]; wherein VH1 and VH2 are an antibody heavy chain variable (VH) domains, wherein CH1 is an antibody CH1 domain, wherein hinge is an antibody hinge domain, wherein CH2-CH3 is an antibody Fc domain, wherein VL1 and VL2 are an antibody light chain variable (VL) domains, and wherein CL is an antibody constant light chain domain; wherein VH1 and VL1 form a first antigen binding site that binds to CD8, and wherein VH2 and VL2 form a second antigen binding site that binds to the activation marker; and wherein the N-terminus of the immunomodulatory polypeptide is fused to the C- terminus of one of the two CH3 domains via a linker.

[0017] In some embodiments according to any of the embodiments described herein, the bispecific antigen binding molecule comprises: an antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

VH1-CH1-hinge-CH2-CH3 [I]; an antibody light chain polypeptide comprising a structure according to formula [II], from N-terminus to C-terminus:

VL1-CL [II]; and an antibody single chain variable fragment (scFv) polypeptide comprising a structure according to formula [V], from N-terminus to C-terminus: VH2-linker-VL2-hinge-CH2-CH3 [V]; wherein VH1 and VH2 are an antibody heavy chain variable (VH) domains, wherein CH1 is an antibody CH1 domain, wherein hinge is an antibody hinge domain, wherein CH2-CH3 is an antibody Fc domain, wherein VL1 and VL2 are an antibody light chain variable (VL) domains, and wherein CL is an antibody constant light chain domain; wherein VH1 and VL1 form a first antigen binding site that binds to CD8 and VH2 and VL2 form a second antigen binding site that binds to the activation marker, or wherein VH1 and VL1 form a first antigen binding site that binds to the activation marker and VH2 and VL2 form a second antigen binding site that binds to CD8; and wherein the N-terminus of the immunomodulatory polypeptide is fused to the C- terminus of one of the two CH3 domains via a linker

[0018] In some embodiments according to any of the embodiments described herein, the bispecific antigen binding molecule comprises: an antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

VL1-CL [II]; and an antibody single domain (VHH) polypeptide comprising a structure according to formula [VI], from N-terminus to C-terminus:

VHH-hinge-CH2-CH3 [VI]; wherein VH1 is an antibody heavy chain variable (VH) domain, wherein CH1 is an antibody CH1 domain, wherein hinge is an antibody hinge domain, wherein CH2-CH3 is an antibody Fc domain, wherein VL1 is an antibody light chain variable (VL) domain, wherein CL is an antibody constant light chain domain, and wherein VHH is an antibody single variable (VHH) domain; wherein VH1 and VL1 form a first antigen binding site that binds to CD8 and VHH forms a second antigen binding site that binds to the activation marker, or wherein VH1 and VL1 form a first antigen binding site that binds to the activation marker and VHH forms a second antigen binding site that binds to CD8; and wherein the N-terminus of the immunomodulatory polypeptide is fused to the C- terminus of one of the two CH3 domains via a linker.

[0019] In some embodiments according to any of the embodiments described herein, one or both of the antibody Fc domains comprise(s) the following amino acid substitutions: L234A, L235A, G237A, and K322A, numbering according to EU index. In some embodiments, a first of the two Fc domains comprises amino acid substitutions Y349C and T366W, and a second of the two Fc domain comprises amino acid substitutions S354C, T366S, L368A and Y407V, numbering according to EU index. In some embodiments, the bispecific antigen binding molecule is fused directly to the immunomodulatory polypeptide. In some embodiments, the bispecific antigen binding molecule is fused to the immunomodulatory polypeptide via a linker. In some embodiments, the linker comprises the sequence (GGGS)xGn (SEQ ID NO:8), (GGGGS)xGn (SEQ ID NO:9), or (GGGGGS)xGn (SEQ. ID NO:10), wherein x=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and wherein n=0, 1, 2 or 3. In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:11).

[0020] In some aspects, provided herein are one or more polynucleotides encoding the fusion protein according to any one of the above embodiments. In some aspects, provided herein are one or more vectors (e.g., expression vector(s)) comprising the one or more polynucleotides of any one of the above embodiments. In some aspects, provided herein is a host cell (e.g., an isolated host cell or cell line) comprising the one or more polynucleotides or vectors of any one of the above embodiments. In some aspects, provided herein are methods of producing a fusion protein, comprising culturing the host cell of any one of the above embodiments under conditions suitable for production of the fusion protein. In some embodiments, the methods further comprise recovering the fusion protein from the host cell. In some aspects, provided herein are pharmaceutical compositions comprising the fusion protein according to any one of the above embodiments and a pharmaceutically acceptable carrier.

[0021] In some aspects, provided herein are the fusion proteins according to any one of the above embodiments for use as a medicament. In some aspects, provided herein are methods of treating cancer comprising administering to an individual with cancer an effective amount of the fusion protein according to any one of the above embodiments or the composition of any one of the above embodiments. In some aspects, provided herein are the fusion proteins according to any one of the above embodiments for use in a method of treating cancer, said method comprising administering to an individual with cancer an effective amount of the fusion protein. In some embodiments, the methods further comprise administering to the individual a T cell therapy, cancer vaccine, chemotherapeutic agent, or immune checkpoint inhibitor (ICI). In some embodiments, the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4. In some embodiments, the T cell therapy comprises a chimeric antigen receptor (CAR)-based T cell therapy, a tumorinfiltrating lymphocyte (TIL)-based therapy, or a therapy with T cells bearing a transduced TCR. In some aspects, provided herein are methods of treating infection (e.g., viral infection) comprising administering to an individual in need thereof an effective amount of the fusion protein according to any one of the above embodiments or the composition of any one of the above embodiments. In some aspects, provided herein is the use of the fusion protein according to any one of the above embodiments or the composition of any one of the above embodiments for the manufacture of a medicament for treating cancer or chronic infection. In some aspects, provided herein are methods of expanding T cells ex vivo comprising contacting one or more T cells ex vivo with an effective amount of the fusion protein according to any one of the above embodiments or the composition of any one of the above embodiments. In some embodiments, the one or more T cells are tumor infiltrating lymphocytes (TILs).

[0022] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the disclosure will become apparent to one of skill in the art. These and other embodiments of the disclosure are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGS. 1A-1D show the amino acid sequence of the following polypeptides: mature IL-2 (FIG. 1A; SEQ ID NO:1), IL-2Rα (FIG. 1B; SEQ ID NO:2), IL-2Rβ ( FIG. 1C; SEQ ID NO:3) and IL-2Rγ (FIG. 1D; SEQ ID NO:4).

[0024] FIGS. 2A & 2B show the amino acid sequence of the following polypeptides: mature IL- 21 (FIG. 2A; SEQ ID NO:5), and IL-21R (FIG. 2B; SEQ ID NO:6).

[0025] FIGS. 3A & 3B show the general mechanism for how fusions with immunomodulatory polypeptides, such as an IL-2Rβγ agonist polypeptide, and CD8 bispecific antigen binding molecules work. FIG. 3A depicts how fusion protein comprising the CD8 bispecific antigen binding molecules that bind to CD8 and an activation marker expressed on CD8+ T cells, such as PD1, selectively activate CD8+ T cells expressing the activation marker, such as PD1, (e.g.

CD8+PD1+ T cells) over immune cells expressing only CD8 or only the activation marker. FIG. 3B compares the activation of CD8+PD1+ T cells by the fusion molecules containing a control antibody, or an antibody binding to only CD8 or only PD1 in comparison to that by a fusion molecule containing a bispecific antibody that binds to CD8 and PD1.

[0026] FIG. 4 shows the amino acid sequence of the wild-type mature IL-2 polypeptide (SEQ ID NO:1) according to EU numbering. "X" denotes the amino acid substituted in the sequence of wild-type mature IL-2 polypeptide for another amino acid to generate the mutant IL-2 polypeptides of the invention.

[0027] FIG. 5 depicts three different fusion molecule formats useful in the present invention. [0028] FIGS. 6Ar6D show the selective targeting of mouse CD8+ T cells expressing PD1 (PD1^highCD8+ T cells) over PD1-CD8+ T cells, PD1^highCD8- Tregs and PD1-CD8-Tregs by the fusion protein comprising the IL-2 mutein, IL-2m1, and the bispecific antibody binding to CD8 and PD1 (xCD8ab1-xPD1ab1-IL2m1), but not by the fusion proteins comprising the IL-2 mutein, IL2m1, and an antibody binding only to CD8 (xCD8ab1-IL2m1) or only to PD1 (xPD1ab1-IL2m1). CD8 antibody xmCD8ab1 was a variant of a previously published anti-mouse CD8 antibody, YTS 105.18, (Shore et al, J Mol Biol. 2006 Apr 28;358(2):347-54). Selective targeting was determined by the induction of phospho STATS as measured by flow cytometry. PD1^highCD8+ T cells and PD1^highCD8- Tregs were isolated from the tumors while PD1-CD8+ T cells and PD1-CD8- Tregs were isolated from spleens of B16.F10 tumor-bearing C57BL6 mice. FIG. 6A depicts the gating strategy for delineating intratumoral PD1^highCD8+ T cells and PD1^highCD8- Tregs. FIG. 6B depicts the activation of STATS in the indicated ceil subsets by xCD8ab1-IL2m1. PD1^highCD8+ T cells and PD1-CD8+ T cells were similarly activated and preferentially over PD1-CD8- and PD1^highCD8-

Tregs. FIG. 6C depicts the activation of STATS in the indicated cell subsets by xPD1ab1-IL2m1. PD1^highCD8+ T cells and PD1^highCD8- Tregs were similarly activated and preferentially over PD1-

CD8+ T cells and PD1-CD8- Tregs. FIG. 6D depicts the activation of STATS in the indicated cell subsets by xCD8ab1-xPD1ab1-IL2m1. CD8-PD1 bispecific antibody of the invention selectively activated PD1^highCD8+T cells over PD1-CD8+ T cells, PD1^highCD8- Tregs, and PD1-CD8-Tregs.

DETAILED DESCRIPTION

Definitions

[0029] As used in this specification and the appended claims, the singular forms "a", "an" and

"the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0030] It is understood that aspects and embodiments of the present disclosure include

"comprising," "consisting," and "consisting essentially of' aspects and embodiments.

[0031] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0032] "Immune cells" as used here are cells of the immune system that react to organisms or other entities that are deemed foreign to the immune system of the host. They protect the host against foreign pathogens, organisms and diseases. Immune cells, also called leukocytes, are involved in both innate and adaptive and immune responses to fight pathogens. Innate immune responses occur immediately upon exposure to pathogens without additional priming or learning processes. Adaptive immune processes require initial priming, and subsequently create memory, which in turn leads to enhanced responsiveness during subsequent encounters with the same pathogen. Innate immune cells include, but are not limited to monocytes, macrophages, dendritic cells, innate lymphoid cells (ILCs) including natural killer (NK) cells, neutrophils, megakaryocytes, eosinophils and basophils. Adaptive immune cells include B and T lymphocytes/cells. T cells subsets include, but are not limited to, alpha beta CD4+ T (naive CD4+, memory CD4+, effector memory CD4+, effector CD4+, regulatory CD4+), and alpha beta CD8+ T (naive CD8+, memory CD8+, effector memory CD8+, effector CD8+). B cell subsets include, but is not limited to, naive B, memory B, and plasma cells. NK T cells and T gamma delta (TyS) cells exhibit properties of both innate and adaptive lymphocytes. In some embodiments, any of the immune cells herein are human cells.

[0033] "T cells" or "T lymphocytes" are immune cells that play a key role in the orchestration of immune responses in health and disease. Two major T cell subsets exist that have unique functions and properties: T cells that express the CD8 antigen (CD8⁺T cells) are cytotoxic or killer T cells that can lyse target cells using the cytotoxic proteins such as granzymes and perforin; and T cells that express the CD4 antigen (CD4⁺ T cells) are helper T cells that are capable of regulating the function of many other immune cell types including that of CD8⁺ T cells, B cells, macrophages etc. Furthermore, CD4⁺ T cells are further subdivided into several subsets such as: T regulatory (Treg) cells that are capable of suppressing the immune response, and T helper 1 (Thl), T helper 2 (Th2), and T helper 17 (Thl7) cells that regulate different types of immune responses by secreting immunomodulatory proteins such as cytokines. T cells recognize their targets via alpha beta T cell receptors that bind to unique antigen-specific motifs and this recognition mechanism is generally required in order to trigger their cytotoxic and cytokine-secreting functions. "Innate lymphocytes" can also exhibit properties of CD8⁺ and CD4⁺ T cells, such as the cytotoxic activity or the secretion of Thl, Th2, and Thl7 cytokines. Some of these innate lymphocyte subsets include NK cells and ILC1, ILC2, and ILC3 cells; and innate-like T cells such as TyS cells; and NK T cells. Typically, these cells can rapidly respond to inflammatory stimuli from infected or injured tissues, such as immunomodulatory cytokines, but unlike alpha beta T cells, they can respond without the need to recognize antigen-specific patterns.

[0034] "Amino acid" as used here refers to naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0035] "Polypeptide" or "protein" as used here refers to a molecule where monomers (amino acids) are linearly linked to one another by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide", and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of a polypeptide may be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. Polypeptides normally have a defined three-dimensional structure, but they do not necessarily have such structure. A polypeptide of the present disclosure may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three- dimensional structure, but rather can adopt many different conformations and are referred to as unfolded. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. The terms "polypeptide" and "protein" also refer to modified polypeptides/proteins wherein the post-expression modification is affected including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[0036] "Residue" as used herein is meant a position in a protein and its associated amino acid identity. For example, Leu 234 (also referred to as Leu234 or L234) is a residue at position 234 in the human antibody IgG1.

[0037] "Wild-type" herein means an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.

[0038] "Substitution" or "mutation" refers to a change to the polypeptide backbone wherein an amino acid occurring in the wild-type sequence of a polypeptide is substituted to another amino acid at the same position in the said polypeptide. In some embodiments, a mutation or mutations are introduced to modify polypeptide's affinity to its receptor thereby altering its activity such that it becomes different from the affinity and activity of the wild-type cognate polypeptide. Mutations can also improve polypeptide's biophysical properties. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.

[0039] "Affinity" or "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). The affinity can generally be represented by the dissociation constant (K_D), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technologies (e.g. BIAcore), BioLayer Interferometry (BLI) technologies (e.g. Octet) and other traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002).

[0040] "Binding" or "specific binding" as used here, refers the ability of a polypeptide or an antigen binding molecule to selectively interact with the receptor for the polypeptide or target antigen, respectively, and this specific interaction can be distinguished from non-targeted or undesired or non-specific interactions.

[0041] "Targeting moiety" and "antigen binding molecule" as used here refers in its broadest sense to a molecule that specifically binds an antigenic determinant. A targeting moiety or antigen binding molecule may be a protein, carbohydrate, lipid, or other chemical compound. It includes, but is not limited to, antibody, antibody fragments (Chames et al, 2009; Chan & Carter, 2010; Leavy, 2010; Holliger & Hudson, 2005), scaffold antigen binding proteins (Gebauer and Skerra, 2009; Stumpp et al, 2008), single domain antibodies (sd Ab), minibodies (Tramontano et al, 1994), the variable domain of heavy chain antibodies (nanobody, VHH), the variable domain of the new antigen receptors (VNAR), carbohydrate binding domains (CBD) (Blake et al, 2006), collagen binding domain (Knight et al, 2000), lectin binding proteins (Tetranectin), collagen binding proteins, adnectin/fibronectin (Lipovsek, 2011 ), a serum transferrin (trans-body), Evibody, Protein A-derived molecule, such as Z-domain of Protein A (Affibody) (Nygren et al, 2008), an A-domain (Avimer/Maxibody), alphabodies (W02010066740), Avimer/Maxibody, designed ankyrin-repeat domains (DARPins) (Stumpp et al, 2008), anticalins (Skerra et al, 2008), a human gamma-crystallin or ubiquitin (Affilin molecules), a kunitz type domain of human protease inhibitors, knottins (Kolmar et al, 2008), linear or constrained peptide with or without fusion to extend half-life e.g. (Fc fusion - Peptibody) (Rentero Rebollo & Heinis, 2013; EP 1144454 B2; Shimamoto et al, 2012; US 7205275 B2) , constrained bicyclic peptides (US 2018/0200378 Al), aptamer, engineered CH2 domains (nanoantibodies; Dimitrov, 2009) ) and engineered CH3 domain "Fcab" domains (Wozniak-Knopp et al, 2010).

[0042] The terms "antibody" and "immunoglobulin" are used interchangeably and herein are used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), antibody fragments and single domain antibody (as described in greater detail herein), so long as they exhibit the desired antigen binding activity.

[0043] "Fab" or "Fab region" as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g. VH-CH1 on one chain and VL-CL on the other).

[0044] "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of an antibody. Examples of formatting for Fv regions include but not limited to: i) non-covalent interacting heterodimer ii) Fabs and iii) single chain Fvs, where the vl and vh domains are linked together to form an scFv.

[0045] Single chain Fv" or "scFv" as used herein is meant a variable heavy domain covalently attached to a variable light domain, generally using a scFv linker as discussed herein, to form a scFv or scFv domain. A scFv domain can be in either orientation from N- to C-terminus (vh- linker-vl or vl-li n ker-vh).

[0046] A single-domain antibody "VHH" or "nanobody" as used herein refers to single monomeric variable antibody domain that bind antigen determinant. The variable antibody domain can be from heavy chain or light chain (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl). "VHH" or "nanobody" may be derived from camelids, llama, and other species which naturally express it. "VHH" or "nanobody" may also be derived from human using recombinant techniques such as VHH or antibody fragment libraries. Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.

[0047] The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The term "bispecific" antibody means that the antibody is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. A bispecific antibody can bind two antigens or two epitopes on the same antigen. In some embodiments, a "bispecific antibody" denotes a single polypeptide chain or multiple (more than two) polypeptide chains connected either through covalent and non-covalent manner comprising two binding domains. Examples of covalent interaction are inter-polypeptide chain disulfide bond, interchain peptide bonds, and chemical bonds. There are a number of exemplary methods on generating bispecific antibody including those in US Patent No. 9.358,286, US Publication 2014/0288275 and WO2014/145806, as well as those depicted and discussed in Kontermann, mAbs 4:2, 182-197 (2012), Spiess et al., Mol. Immunol. 2015, Brinkmann & Kontermann mAbs 2017 and Godar et al, Expert Opinion on Therapeutic Patents 2018.

[0048] In some embodiments, the recombinant bispecific antibodies disclosed herein can be very roughly classified in two categories, namely i) formats resulting from the combination of variable regions only and ii) formats combining variable regions with Fc domains. representatives of the first category are tandem scFv (taFv), diabodies (Db), DART, single-chain diabodies (scDbs), Fab-Fc, tandem Fab, Dual variable region Fab and tandem dAb/VHH. The two variable regions can be linked together via covalent bonds or non-covalent interaction. Non- covalent interaction may involve the use of heterodimerization modules such as leucine zipper, dock-and-lock methods of using regulatory subunit of cAMP-dependent protein kinase (PKA) and the anchoring domains of A kinase anchor proteins (AKAPs) or knob-into-holes CH3 domain (U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001)) to pair up the variable regions.

[0049] In some embodiments, bispecific antibodies are generated on the natural immunoglobulin architecture containing two pairs of heavy chain and light chain combination with each pair having distinct binding specificity. Homodimerization of the two heavy chains in an IgG is mediated by the CH3 interaction. To promote heterodimeric formation, genetic modifications are introduced to the two respective CH3 regions. There heterodimerization mutations often involve steric repulsion, charge steering interaction, or interchain disulfide bond formation. Exemplary and non-limiting Fc modifications to promote heterodimerization include the following:

[0050] In some embodiments, said first and second Fc domains of the fusion protein contain the following Fc mutations to decrease effector function according to EU numbering: L234A, L235A, G237A, and K322A. In some embodiments, said first and second Fc domains of the fusion protein contain the following Fc mutations to decrease effector function according to EU numbering: L234A, L235A, G237A, and K322A. In some embodiments, said first and second Fc domains of the fusion protein contain the following amino acid substitutions to facilitate heterodimeric formation: Y349C/T366W (knob) and S354C, T366S, L368A and Y407V (hole). [0051] In some embodiments, bispecific antibody can be generated by post-production assembly from half-antibodies, thereby solving the issues of heavy and light chain mispairing. These antibodies often contain modification to favor heterodimerization of half-antibodies.

Exemplary systems include but not limited to the knob-into-hole, IgG1 (EEE - RRR), lgG2 (EEE - RRRR) (Strop et al. J Mol Biol (2012)) and DuoBody (F405L-K409R), listed in Table 5. In such case, half-antibody is individually produced in separate cell line and purified. The purified antibodies were then subjected to mild reduction to obtain half-antibodies, which were then assembled into bispecific antibodies. Heterodimeric bispecific antibody was then purified from the mixture using conventional purifications methods.

[0052] In some embodiments, strategies on bispecific antibody generation that do not rely on the preferential chain pairing can also be employed. These strategies typically involve introducing genetic modification on the antibody in such a manner that the heterodimer will have distinct biochemical or biophysical properties from the homodimers; thus the postassembled or expressed heterodimer can be selectively purified from the homodimers. One example was to introduce H435R/Y436F in IgG1 CH3 domain to abolish the Fc binding to protein A resin and then co-express the H435R/Y436F variant with a wildtype Fc. The resulting homodimeric antibodies containing two copies of H435R/Y436F cannot bind to the Protein A column, while heterodimeric antibody comprising one copy of H435R/Y436F mutation will have a decreased affinity for protein A as compared to the strong interaction from homodimeric wildtype antibody (Tustian et al Mabs 2016). Other examples include kappa/lambda antibody (Fischer et al., Nature Communication 2015) and introduction of differential charges (E357Q, S267K or N208D/Q295E/N384D/Q418E/N421D) on the respective chains (US 2018/0142040 Al; (Strop et al. J Mol Biol (2012)).

[0053] In some embodiments, bispecific antibody can be generated via fusion of an additional binding site to either the heavy or light chain of an immunoglobulin. Examples of the additional binding site include but not limited to variable regions, scFv, Fab, VHH, and peptide.

[0054] In some embodiments, antibodies (immunoglobulins) refer to a protein having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native immunoglobulins of the IgG class are heterotetra meric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C- terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and described generally, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). Antibodies (immunoglobulins) are assigned to different classes, depending on the amino acid sequences of the heavy chain constant domains. There are five major classes of antibodies: a (IgA), δ (IgD), ∈ (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g. γl (IgG 1), γ2 (lgG2), γ3 (lgG3), γ4 (lgG4), α1 (IgAl) and α2 (lgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

[0055] "Fc" or "Fc region" or "Fc domain" as used herein refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. An Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally, all or a portion of the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain and in some cases, inclusive of the hinge. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The "hinge" region usually extends from amino acid residue at about position 216 to amino acid residue at about position 230. The hinge region herein may be a native hinge domain or variant hinge domain. The "CH2 domain" of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The "CH3 domain" comprises the stretch of residues C- terminal to a CH2 domain in an Fc region, from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG. The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced "protuberance" ("knob") in one chain thereof and a corresponding introduced "cavity" ("hole") in the other chain thereof; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Thus, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An "Fc fragment" in this context may contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods, generally based on size (e.g. non-denaturing chromatography, size exclusion chromatography, etc.). Human IgG Fc domains are of particular use in the present disclosure, and can be the Fc domain from human IgG1, lgG2 or lgG4.

[0056] A "variant Fc domain" or "Fc variant" or "variant Fc" contains amino acid modifications (e.g. substitution, addition, and deletion) as compared to a parental Fc domain.

The term also includes naturally occurring allelic variants of the Fc region of an immunoglobulin. In general, variant Fc domains have at least about 80, 85, 90, 95, 97, 98 or 99 percent identity to the corresponding parental human IgG Fc domain (using the identity algorithms discussed below, with one embodiment utilizing the BLAST algorithm as is known in the art, using default parameters). Alternatively, the variant Fc domains can have from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications as compared to the parental Fc domain. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Additionally, as discussed herein, the variant Fc domains herein still retain the ability to form a dimer with another Fc domain as measured using known techniques as described herein, such as non-denaturing gel electrophoresis. [0057] "Fc gamma receptor", "FcγR" or "Fc gamma R" as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRla, FcγRIb, and FcγRIc; FcγRI I (CD32), including isoforms FcγRI la (including allotypes H131 and R131), FcγRllb (including FcγRllb-1 and FcγRI lb-2), and FcγRI Ic; and FcγRIII (CD16), including isoforms FcγRI I la (including allotypes V158 and F158) and FcγRI 11 b (including allotypes Fey Rl I b- NA1 and Fey Rl I b-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), Fey Rl I (CD32), FcγRIII (CD16), and FcγRI 11-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes.

[0058] "Epitope" as used herein refers to a determinant capable of specific binding to the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the antigen binding peptide (in other words, the amino acid residue is within the footprint of the antigen binding peptide). Epitopes may be either conformational or linear. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example "binning".

[0059] "Linker" as used herein refers to a molecule that connect two polypeptide chains. Linker can be a polypeptide linker or a synthetic chemical linker (for example, see disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linkers is not particularly limited and can be selected according to the purpose by those skilled in the art. Polypeptide linker comprises one or more amino acids. In some embodiments, the polypeptide linker is a peptide with a length of at least 5 amino acids, in some embodiments with a length of 5 to 100, or 10 to 50 amino acids. In one embodiment, said peptide linker is G, S, GS, SG, SGG, GGS, and GSG (with G=glycine and S=serine). In another embodiment, said peptide linker is (GGGS)xGn (SEQ ID NO:8) or (GGGGS)xGn (SEQ ID NO:9) or (GGGGGS)xGn (SEQ ID NO:10) with x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n=0, 1, 2 or 3. In some embodiments, said linker is (GGGGS)xGn with x=2,3, or 4 and n=0 (SEQ ID NO:12); in some embodiments the said linker is (GGGGS)xGn with x=3 and n=0 (SEQ ID NO:13). In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:11). Synthetic chemical linkers include crosslinking agents that are routinely used to crosslink peptides, for example, N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobisfsuccinimidyl propionate) (DSP), dithiobisfsuccinimidyl propionate) (DTSSP), ethylene glycol bisfsucci nimidy I succinate) (EGS), ethylene glycol bisfsulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).

[0060] Percent (%) amino acid sequence identity" with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. One particular program is the ALIGN-2 program outlined at paragraphs [0279] to [0280] of US Pub. No. 20160244525, hereby incorporated by reference.

[0061] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA) encoding the polypeptides of the present disclosure. A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g. an amide bond, such as found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g. DNA or RNA fragments, present in a polynucleotide. In some aspects, one or more vectors (particularly expression vectors) comprising such nucleic acids are provided. In one aspect, a method for making a polypeptide of the present disclosure is provided, wherein the methods comprises culturing a host cell comprising a nucleic acid encoding the polypeptide under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell. "Recombinant" means the proteins are generated using recombinant nucleic acid techniques in exogeneous host cells. Recombinantly produced proteins expressed in host cells are considered isolated for the purpose of the present disclosure, as are native or recombinant proteins which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0062] "Isolated," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Typically, an isolated polypeptide will be purified by at least one purification step. There is no required level of purity; "purification" or "purified" refers to increase of the target protein concentration relative to the concentration of contaminants in a composition as compared to the starting material. An "isolated protein," as used herein refers to a target protein which is substantially free of other proteins having different binding specificities.

[0063] The terms "cancer" refers the physiological condition in mammals that is typically characterized by unregulated and abnormal cell growth with the potential to invade or spread to other parts of the body. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include lung cancer, small-cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, squamous cell cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, thyroid cancer, uterine cancer, , gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, endometrial carcinoma, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, vulval cancer, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, bladder cancer, liver cancer, hepatoma, hepatocellular cancer, cervical cancer, salivary gland carcinoma, biliay cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

EXAMPLES

Example 1: Activation of mouse immune cells from spleens and tumors in pSTAT5 assay

[0064] Splenocytes were isolated from spleens of B6 mice by placing a spleen onto a 70 mM strainer and using a plunger to wash the cells with PBS through the strainer. Red blood cells were lysed with ACK lysis buffer and cells resuspended at 20x10⁶/ml of RPMI media. Cells were plated in U-bottom plates at 50 ml per well at 0.5-1 x 10⁶ cells per well.

[0065] Tumors from mice implanted with B16.F10 cells (ATCC, CRL-6475) were digested to single cells using Mouse Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730) in Miltenyi Gentle MACS C tubes according to manufacturer’s protocol. Isolated cells from multiple tumors were pooled and counted and CD45+ cells isolated using LS columns (Miltenyi) according to manufacturer’s protocol. Cells were plated in U-bottom plates at 50 μl per well.

[0066] IL-2 fusion proteins and control proteins were added to cells (50 μl as 2x stimulus) for 30min at 37°C. PD1 antibody (RMP1-30 done) was added directly to each well on ice and incubated for 10-15 min. Spleen and tumor cells were fixed with 8% PFA (4% final). Cells were washed 2x with PBS-2% FBS and resuspended in 75 μl Phosflow Perm buffer III buffer and incubated for 1 hr at 4°C or overnight at -20°C. Cells were washed 3x with PBS-2% FBS and stained in 50 μl of FACS buffer containing antibodies against CDS (17A2), CD4 (GK1.5), CD8a (53-6.7), CD8b (YTS156.7.7), CD25 (704), and pSTATS (clone 47). Samples were washed 2x and analyzed on a flow cytometer.

[0067] FIG. 6 shows the selective targeting of mouse CD8+ T cells expressing PD1

(PD1^highCD8+ T cells) over PD1-CD8+ T cells, PD1^CD8- Tregs and PD1-CD8-Tregs by the fusion protein comprising the IL-2 mutein, IL-2m1, and the bispecific antibody binding to CD8 and PD1 (xCD8ab1-xPD1ab1-IL2m1), but not by the fusion proteins comprising the IL-2 mutein, IL2m1, and an antibody binding only to CD8 (xCD8ab1-IL2m1) or only to PD1 (xPD1ab1-IL2m1). CD8 antibody xmCD8ab1 was a variant of a previously published anti-mouse CD8 antibody, YTS 105.18, (Shore et al, J Mol Biol. 2006 Apr 28;358(2):347-54). Selective targeting was determined by the induction of phospho STATS as measured by flow cytometry. PD1^highCD8+ T cells and PD1^highCD8- Tregs were isolated from the tumors while PD1-CD8+ T cells and PD1-CD8- Tregs were isolated from spleens of B16.F10 tumor-bearing C57BL6 mice. FIG. 6A depicts the gating strategy for delineating intratumoral PD1^highCD8+ T cells and PD1^highCD8- Tregs. FIG. 6B depicts the activation of STATS in the indicated cell subsets by xCD8ab1-IL2m1. PD1^highCD8+ T cells and PD1-CD8+ T cells were similarly activated and preferentially over PD1-CD8- and PD1^highCD8- Tregs. FIG. 6C depicts the activation of STATS in the indicated cell subsets by xPD1ab1-IL2m1. PD1^highCD8+ T cells and PD1^highCD8- Tregs were similarly activated and preferentially over PD1- CD8+ T cells and PD1-CD8- Tregs. FIG. 6D depicts the activation of STATS in the indicated cell subsets byxCD8ab1-xPD1ab1-IL2m1. CD8-PD1 bispecific antibody of the invention selectively activated PD1^highCD8+ T cells over PD1-CD8+ T cells, PD1^highCD8- Tregs, and PD1-CD8-Tregs.

Claims

What is claimed is: 1. A fusion protein comprising:

(a) a bispecific antigen binding molecule, wherein the bispecific antigen binding molecule comprises:

(i) a first antigen binding domain that binds to CD8, and

(ii) a second antigen binding domain that binds to an activation marker expressed on CD8+ T cells; and

(b) an immunomodulatory polypeptide; wherein the bispecific antigen binding molecule is fused to the immunomodulatory polypeptide; wherein the fusion protein selectively activates CD8+ T cells expressing the activation marker over immune cells expressing only CD8 or only the activation marker; and wherein the CD8+ T cells further express a receptor for the immunomodulatory polypeptide, and the fusion protein activates the immune cells by activation of the receptor via the immunomodulatory polypeptide.

2. The fusion protein of claim 1, wherein the activation marker is selected from the group consisting of PD1, CD137, CD39, CD69, CD103, LAG3, and TIM3.

3. The fusion protein of claim 1 or claim 2, wherein the immunomodulatory polypeptide is selected from the group consisting of IL-2, IL-7, IL-10, IL-15, IL-18, IL-21, and mutants thereof, wherein the mutants of IL-2, IL-7, IL-10, IL-15, IL-18, or IL-21 are capable of activating signaling via the corresponding receptor.

4. The fusion protein of claim 1 or claim 2, wherein said immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rα polypeptide comprising the amino acid sequence of SEQ ID NO:2, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL- 2Rα polypeptide.

5. The fusion protein of claim 4, wherein said immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:3, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL-2Rβ polypeptide.

6. The fusion protein of claim 4 or claim 5, wherein said mutant immunomodulatory polypeptide is a mutant IL-2 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO:4, compared to binding affinity of a wild-type IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL- 2Rγ polypeptide.

7. The fusion protein of claim 1 or claim 2, wherein said immunomodulatory polypeptide is:

(a) an IL-2Rβγ agonist polypeptide that binds to and/or activates an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:3; and/or

(b) an IL-2RβV polypeptide agonist polypeptide that binds to and/or activates an IL-2Rγ polypeptide comprising the amino acid sequence of SEQ ID NO:4.

8. The fusion protein of any one of claims 4-6, wherein said mutant IL-2 polypeptide comprises one or more amino acid substitutions relative to a wild-type IL-2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:1, and wherein the one or more amino acid substitution(s) are at one or more position(s) selected from the group consisting of: Qll, E15, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, N88, V91, 192, T123, Q126, S127, 1129, S130, according to the wild-type IL-2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:1.

9. The fusion protein of any one of claims 4-6, wherein said mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:1 with one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO:1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E62Q; and F42A, E62Q, and Q126S.

10. The fusion protein of claim 9, wherein the mutant IL-2 polypeptide comprises a further amino acid substitution relative to SEQ ID NO:1 at position C125.

11. The fusion protein of claim 10, wherein the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:1 with one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 1): R38E, F42A, and C125A; R38D, F42A , and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A , C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A;

F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; and F42A, C125A and Q126S.

12. The fusion protein of any one of claims 4-11, wherein the mutant IL-2 polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID N0:7).

13. The fusion protein of claim 1 or claim 2, wherein said immunomodulatory polypeptide is a mutant IL-21 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-21R polypeptide comprising the amino acid sequence of SEQ ID NO:6, compared to binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO:5 to the IL- 21R polypeptide.

14. The fusion protein of claim 13, wherein said immunomodulatory polypeptide is a mutant IL-21 polypeptide that exhibits reduced binding affinity by 50% or more to an IL-2Rg polypeptide comprising the amino acid sequence of SEQ ID NO:4, compared to binding affinity of a wild-type IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO:5 to the IL-2Rg polypeptide.

15. The fusion protein of any one of claims 1-14, wherein the bispecific antigen binding molecule comprises: a first antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

VL1-CL [II]; a second antibody heavy chain polypeptide comprising a structure according to formula [III], from N-terminus to C-terminus:

[IV], from N-terminus to C-terminus: VL2-CL [IV]; wherein VH1 and VH2 are an antibody heavy chain variable (VH) domains, wherein CH1 is an antibody CH1 domain, wherein hinge is an antibody hinge domain, wherein CH2-CH3 is an antibody Fc domain, wherein VL1 and VL2 are an antibody light chain variable (VL) domains, and wherein CL is an antibody constant light chain domain; wherein VH1 and VL1 form a first antigen binding site that binds to CD8, and wherein VH2 and VL2 form a second antigen binding site that binds to the activation marker; and wherein the N-terminus of the immunomodulatory polypeptide is fused to the C- terminus of one of the two CH3 domains via a linker.

16. The fusion protein of any one of claims 1-14, wherein the bispecific antigen binding molecule comprises: an antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

VL1-CL [II]; and an antibody single chain variable fragment (scFv) polypeptide comprising a structure according to formula [V], from N-terminus to C-terminus:

VH2-linker-VL2-hinge-CH2-CH3 [V]; wherein VH1 and VH2 are an antibody heavy chain variable (VH) domains, wherein CH1 is an antibody CH1 domain, wherein hinge is an antibody hinge domain, wherein CH2-CH3 is an antibody Fc domain, wherein VL1 and VL2 are an antibody light chain variable (VL) domains, and wherein CL is an antibody constant light chain domain; wherein VH1 and VL1 form a first antigen binding site that binds to CD8 and VH2 and

VL2 form a second antigen binding site that binds to the activation marker, or wherein VH1 and

VL1 form a first antigen binding site that binds to the activation marker and VH2 and VL2 form a second antigen binding site that binds to CD8; and wherein the N-terminus of the immunomodulatory polypeptide is fused to the C- terminus of one of the two CH3 domains via a linker.

17. The fusion protein of any one of claims 1-14, wherein the bispecific antigen binding molecule comprises: an antibody heavy chain polypeptide comprising a structure according to formula [I], from N-terminus to C-terminus:

18. The fusion protein of any one of claims 15-17, wherein one or both of the antibody Fc domains comprise(s) the following amino acid substitutions: L234A, L235A, G237A, and K322A, numbering according to EU index.

19. The fusion protein of any one of claims 15-18, wherein a first of the two Fc domains comprises amino acid substitutions Y349C and T366W, and a second of the two Fc domain comprises amino acid substitutions S354C, T366S, L368A and Y407V, numbering according to EU index.

20. The fusion protein of any one of claims 1-14, wherein the bispecific antigen binding molecule is fused to the immunomodulatory polypeptide via a linker.

21. The fusion protein of any one of claims 15-20, wherein the linker comprises the sequence

(GGGS)xGn (SEQ ID NO:8), (GGGGS)xGn (SEQ ID NO:9), or (GGGGGS)xGn (SEQ ID NQ:10), wherein x=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and wherein n=0, 1, 2 or 3.

22. The fusion protein of any one of claims 15-20, wherein the linker comprises the sequence

GGGGSGGGGSGGGGS (SEQ ID NO:11).

23. One or more polynucleotides encoding the fusion protein according to any one of claims 1-22.

24. One or more vectors comprising the one or more polynucleotides of claim 23.

25. The one or more vectors of claim 24, wherein the vector(s) are expression vector(s).

26. An isolated host cell comprising the one or more polynucleotides or vectors of any one of claims 20-25.

27. A method of producing a fusion protein, comprising culturing the host cell of claim 26 under conditions suitable for production of the fusion protein.

28. The method of claim 27, further comprising recovering the fusion protein from the host cell.

29. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-

22 and a pharmaceutically acceptable carrier.

30. The fusion protein according to any one of claims 1-22 for use as a medicament.

31. A method of treating cancer comprising administering to an individual with cancer an effective amount of the fusion protein according to any one of claims 1-22 or the composition of claim

29.

32. The method of claim 31, further comprising administering to the individual a T cell therapy, cancer vaccine, chemotherapeutic agent, or immune checkpoint inhibitor (ICI).

33. The method of claim 32, wherein the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4.

34. The method of claim 32, wherein the T cell therapy comprises a chimeric antigen receptor

(CAR)-based T cell therapy, a tumor-infiltrating lymphocyte (TIL)-based therapy, or a therapy with T cells bearing a transduced TCR.

35. The fusion protein according to any one of claims 1-22 for use in a method of treating cancer, said method comprising administering to an individual with cancer an effective amount of the fusion protein.

36. A method of treating infection comprising administering to an individual in need thereof an effective amount of the fusion protein according to any one of claims 1-22 or the composition of claim 29.

37. The method of claim 36, wherein the infection is a viral infection.

38. Use of the fusion protein according to any one of claims 1-22 for the manufacture of a medicament for treating cancer or chronic infection.

39. A method of expanding T cells ex vivo comprising contacting one or more T cells ex vivo with an effective amount of the fusion protein according to any one of claims 1-22 or the composition of claim 29.

40. The method of claim 39, wherein the one or more T cells are tumor infiltrating lymphocytes

(TILs).