Compositions of engineered cytokine expressing cells and methods of use thereof

Abstract

Provided herein is an immune cell expressing a recombinant IL-2 or IL-2 mutein, wherein the immune cell is a natural killer cells (e.g., a cord blood derived NK (CB-NK) cell) or a γδ T cell, and wherein the IL-2 mutein has reduced binding to IL2 receptor (IL2R) (e.g., IL2Rβ). These immune cells can also be used in conjunction with cells genetically engineered with chimeric antigen receptors (CARs). Also provided are methods of modulating immune function by administering said immune cell, and methods of treating cancer, for example, acute myeloid leukemia.

Description

ATTORNEY DOCKET NO. MIL-052WO1COMPOSITIONS OF ENGINEERED CYTOKINE EXPRESSING CELLS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of U. S. Provisional Application No: 63 / 712,126, filed on October 25, 2024; the entirety of the application is incorporated herein by reference.CROSS REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 23, 2025, is named MIL-052WO1_SL.xml and is 80,013 bytes in size.BACKGROUND

[0003] Immunotherapy with immune cells which are genetically modified to express chimeric antigen receptors (CARs), is a promising approach to improve outcomes for cancer patients. Physiological T cell activation requires three distinct signals for acquisition of effector function and formation of immunological memory. Signal 1 (activation) occurs via CD3ζ signal transduction following T cell receptor (TCR)-mediated antigen recognition. Although activated CAR T cells produce cytokines, such as interleukin-2 (IL-2), production decreases after repeated exposure to tumor cells, and some cytokines that are important for T cell effector function, such as IL-12 and IL-15, are either produced at low levels or not at all by T cells. The common gamma chain family of cytokines - IL-2, IL-4, IL-7, IL-9, IL- 15, and IL-21 - play critical roles in T cell differentiation, proliferation, and homeostasis. IL-2 and IL- 15 cytokines share the common gamma chain (yc) and the IL-2RP chain for signal transduction and private receptors (IL-2Ra and IL-15Ra) for receptor specificity. The receptors for other cytokines include the common gamma chain (yc) and a private receptor chain for receptor specificity (IL-4Ra, IL-7Ra, IL-9Ra, IL-21R).

[0004] It is known that transgenic cytokines not only enhance the antitumor activity of CAR T cells but also modulate other cells within the tumor microenvironment and are able to induce or enhance endogenous tumor-specific immune responses. IL-2 is a 15 kDa cytokine predominantly secreted by activated T cells and represents a key player in the activation, proliferation and survival of T cells. Human IL-2 is synthesized as a 153-amino-acidATTORNEY DOCKET NO. MIL-052WO1precursor polypeptide and then processed to mature IL-2. Mature human IL-2 is a four-a-helix bundle glycoprotein that consists of 133 amino acids. Initially called T cell growth factor, IL-2 stimulates proliferation and enhances function of other T cells, natural killer (NK) cells and B-cells.

[0005] IL-2 has been shown to enhance the proliferation and cytotoxicity of NK cells or gamma delta T cells in vitro and in vivo. In vitro studies demonstrate that the combination of IL-2 and IL-12 has a synergistic effect in promoting IFN-y production by T and NK cells, and the cytotoxic activity of NK cells or gamma delta T cells. Despite the wealth of knowledge about IL-2 (including IL-2 superagonists), there remains a need in the art for better combination therapies to treat cancer.SUMMARY OF INVENTION

[0006] The present invention relates to immune cells comprising a membrane-bound, attenuated cytokine signaling molecule. The membrane-bound attenuated cytokine signal is configured to mediate cis-activation within the engineered immune effector cells (e.g., CAR or TCR expressing immune cells) while reducing or preventing systemic cytokine exposure and activation of bystander immune effector cells. Without being bound by any theory, the fine-tuning the affinity of the attenuated cytokine allows for an optimal balance between therapeutic efficacy and safety of the cell therapy products.

[0007] In yet another aspect, the present invention provides, among other things, an immune cell expressing a recombinant IL-2 mutein, wherein the IL-2 mutein has reduced binding to IL2 receptor (IL2R). In some embodiments, the immune cell is a Natural killer cells (e.g., a cord blood derived NK (CB-NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a y5 T cell (e.g., a blood y5 T cell or a skin derived y5 T cell). In some embodiments, the IL-2 mutein has reduced binding to IL2Rp. In some embodiments, the IL-2 mutein has reduced binding to IL-2Ry, or IL-2Ra.

[0008] In some embodiments, the recombinant IL-2 mutein comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, R38E, F42A, L80F, R81D, L85V, I86V, N88R, N88T, V91T, I92F, Q126A, Q126C, Q126E, Q126H, Q126K, and Q126T, numbered in accordance with wild-type human IL-2.

[0009] In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 11. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 1. In some embodiments, theATTORNEY DOCKET NO. MIL-052WO1recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 3. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 4. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 5. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 6. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 7. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 8. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 9. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 10. In some embodiments, the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to SEQ ID NO: 11.

[0010] In some embodiments, the recombinant IL-2 mutein is a soluble form of the recombinant IL-2 mutein.

[0011] In some embodiments, the recombinant IL-2 mutein is a membrane bound form of the recombinant IL-2 mutein.

[0012] In some embodiments, the membrane bound form of the recombinant IL-2 mutein comprises a hinge and a transmembrane domain selected from CD4, CD8, CD28, and B7. In some embodiments, the membrane bound form of the recombinant IL-2 mutein comprises a hinge and a transmembrane domain from CD4. In some embodiments, the membrane bound form of the recombinant IL-2 mutein comprises a hinge and a transmembrane domain from CD8. In some embodiments, the membrane bound form of the recombinant IL-2 mutein comprises a hinge and a transmembrane domain from CD28. In some embodiments, the membrane bound form of the recombinant IL-2 mutein comprises a hinge and a transmembrane domain from B7.

[0013] In some embodiments, the membrane bound form of the recombinant IL-2 mutein further comprises a CTLA4 intracellular domain.

[0014] In some embodiments, the IL-2 mutein comprises a signal peptide. In some embodiments, the IL-2 mutein comprises signal peptide that may further reduce its surface expression. In some embodiments, the signal peptide is a signal peptide from IL- 15. As non-ATTORNEY DOCKET NO. MIL-052WO1limiting examples, the signal peptide comprises the sequence of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 74).

[0015] In some embodiments, the immune cell further expresses a chimeric antigen receptor (CAR).

[0016] In some embodiments, the CAR comprises an extracellular binding domain, a transmembrane domain, and at least one intracellular signaling domain.

[0017] In some embodiments, the extracellular antigen binding domain is an IgA antibody, IgG antibody, IgE antibody, IgM antibody, bi- or multi- specific antibody, Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fd’ fragment, Fd fragment, isolated CDRs or sets thereof; single-chain variable fragment (scFv), polypeptide-Fc fusion, single domain antibody (sdAb), camelid antibody; masked antibody, Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain, Tandem diabody, VHHs, Anticalin, Nanobody, humabody, minibodies, BiTE, ankyrin repeat protein, DARPIN, Avimer, DART, TCR-like antibody, Adnectin, Affilin, Trans-body; Affibody, TrimerX, MicroProtein, Fynomer, Centyrin; and KALBITOR; optionally wherein the GCC binding agent is a single domain antibody (sdAb) or a heavy chain only antibody, or fragment thereof. In some embodiments, the extracellular antigen binding domain is an IgA antibody. In some embodiments, the extracellular antigen binding domain is an IgG antibody. In some embodiments, the extracellular antigen binding domain is an IgE antibody. In some embodiments, the extracellular antigen binding domain is an IgM antibody. In some embodiments, the extracellular antigen binding domain is a bi- or multi- specific antibody. In some embodiments, the extracellular antigen binding domain is a bispecific antibody. In some embodiments, the extracellular antigen binding domain is a multispecific antibody. In some embodiments, the extracellular antigen binding domain is a Fab fragment. In some embodiments, the extracellular antigen binding domain is a Fab’ fragment. In some embodiments, the extracellular antigen binding domain is a F(ab’)2 fragment. In some embodiments, the extracellular antigen binding domain is a Fd’ fragment. In some embodiments, the extracellular antigen binding domain is a Fd fragment. In some embodiments, the extracellular antigen binding domain is isolated CDRs or sets thereof. In some embodiments, the extracellular antigen binding domain is a single chain variable fragment (scFv). In some embodiments, the extracellular antigen binding domain is a polypeptide-Fc fusion. In some embodiments, the extracellular antigen binding domain is a single domain antibody (sdAb). In some embodiments, the extracellular antigen binding domain is a camelid antibody. In some embodiments, the extracellular antigen binding domain is a masked antibody. In some embodiments, the extracellular antigen bindingATTORNEY DOCKET NO. MIL-052WO1domain is a Small Modular ImmunoPharmaceuticals (“SMIPsTM”). In some embodiments, the extracellular antigen binding domain is a single chain. In some embodiments, the extracellular antigen binding domain is a Tandem antibody. In some embodiments, the extracellular antigen binding domain is a VHH. In some embodiments, the extracellular antigen binding domain is an Anticalin. In some embodiments, the extracellular antigen binding domain is a Nanobody. In some embodiments, the extracellular antigen binding domain is a humabody. In some embodiments, the extracellular antigen binding domain is minibodies. In some embodiments, the extracellular antigen binding domain is BiTE. In some embodiments, the extracellular antigen binding domain is an ankyrin repeat protein. In some embodiments, the extracellular antigen binding domain is a DARPIN. In some embodiments, the extracellular antigen binding domain is an Avimer. In some embodiments, the extracellular antigen binding domain is a DART. In some embodiments, the extracellular antigen binding domain is a TCR-like antibody. In some embodiments, the extracellular antigen binding domain is an Adnectin. In some embodiments, the extracellular antigen binding domain is an Affilin. In some embodiments, the extracellular antigen binding domain is a Trans-body. In some embodiments, the extracellular antigen binding domain is a TrimerX. In some embodiments, the extracellular antigen binding domain is a MicroProtein. In some embodiments, the extracellular antigen binding domain is a Fynomer. In some embodiments, the extracellular antigen binding domain is a Centyrin. In some embodiments, the extracellular antigen binding domain is a KALBITOR. In optional embodiments, the GCC binding agent is a single domain antibody (sdAb) or a heavy chain only antibody, or fragment thereof.

[0018] In some embodiments, the extracellular antigen domain binds a tumor associated antigen selected from 4- IBB, 5 AC, 5T4, A2aR, activin receptor-like kinase 1, AGS-22M6, AKAP4, alpha-fetoprotein, angiopoietin 2, B7-H3, BAFF, BAGE, BCR-ABL, BORIS, CA-125, CA19-9, C242 antigen, carbonic anhydrase 9 (CA-IX), CCR4, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD28, CD30 (TNFRSF8), CD33, CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 v6, CD51, CD56, CD70, CD71, CD73, CD74, CD79B, CD80, CD137, CD140a, CD152, CD200, CD221, CD274, CEA, ch4D5, CLDN18.2, CS1, CSF1R, CTLA-4, C-X-C chemokine receptor type 4, DLL4, DR5, EBAG9, EGF, EGFR, EGFL7, EpCAM, ERBB2, ERBB3, FAP, fibronectin extra domain-B, folate receptor 1, folate receptor alpha, folate hydrolase, Frizzled receptor, GAGE, GD2 ganglioside, GD3 ganglioside, glioma, glypican 3, GP MB, gp100, guanylate cyclase 2C (GUCY2C), HER1, HER2 / neu, HER3, HGF, HHGFR, histone complex, HLA-DR, human scatter factor receptorATTORNEY DOCKET NO. MIL-052WO1kinase, HPV-16, HSP105, IDH1, IDO1, IGF-I, IGF-1 receptor, ILGF2, IL-6, IL-13, integrin avP3, integrin a5 1, KIR, LAG-3, Lewis-Y antigen, LY6K, MAGE-1, MAGE- A3, MAGE-C2, MAGE-D4, MAPG, MART-1, Melan-A, MET, MCP-1, mesothelin, MIF, MSLN (Mesothelin), MS4A1, mucin CanAg, MUC1, MUC4, MUC16, NG2, N-glycolylneuraminic acid, Notch receptor PD-1, NY-ESO-1, OCAA, PAP, PDGF-Ra, PDCD1, PD1, PD-L1, phosphate-sodium co-transporter, phosphatidylserine, PRAME, PSA, RANKL, RON, R0R1, SDC1, Sialyl-Tn, SLAMF7, SPAG-9, SSX1, STEAP1, survivin, TAG- 72, telomerase, TEM1, tenascin C, TGF-P, TFM-3, TLR, TAM, TFM-3, TRAIL-R2, TRAIL-R1, TWEAK receptor, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor antigen CTAA16.88, TYRP1 (glycoprotein 75), VEGF-A, VEGFR2, VEGFR-1, vimentin, VISTA, WT1, and XAGE-lb. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen 4-IBB. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen 5 AC. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen 5T4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen A2aR. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen activin receptor-like kinase 1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen AGS-22M6. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen AKAP4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen alpha-fetoprotein. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen angiopoietin 2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen B7-H3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen BAFF. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen BAGE. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen BCR-ABL. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen BORIS. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CA-125. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CAI 9-9. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen C242 antigen. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen carbonic anhydrase 9 (CA-IX). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigenATTORNEY DOCKET NO. MIL-052WO1CCR4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD 19. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD20. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD22. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD23 (IgE receptor). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD24. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD28. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD30 (TNFRSF8). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD33. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD37. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD38 (cyclic ADP ribose hydrolase). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD40. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD44 v6. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD51. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD56. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD70. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD71. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD73. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD74. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD79B. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD80. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD137. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD 140a. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD 152. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD200. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD221. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CD274. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CEA. In some embodiments, the extracellular antigen binding domain binds the tumorATTORNEY DOCKET NO. MIL-052WO1associated antigen ch4D5. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CLDN18.2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CS1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CSF1R. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen CTLA-4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen C-X-C chemokine receptor type 4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen DLL4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen DR5. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen EBAG9. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen EGF. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen EGFR. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen EGFL7. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen EpCAM. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen ERBB2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen ERBB3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen FAP. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen fibronectin extra domain-B. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen folate receptor 1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen folate receptor alpha. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen folate hydrolase. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen Frizzled receptor. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen GAGE. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen GD2 ganglioside. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen GD3 ganglioside. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen glioma. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen glypican 3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen GP MB. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen gp100. In some embodiments, theATTORNEY DOCKET NO. MIL-052WO1extracellular antigen binding domain binds the tumor associated antigen guanylate cyclase 2C (GUCY2C). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HER1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HER2 / neu. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HER3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HGF. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HHGFR. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen histone complex. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HLA-DR. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen human scatter factor receptor kinase. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HPV-16. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen HSP105. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IDH1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IDO1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IGF-I. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IGF-1 receptor. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen ILGF2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IL-6. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen IL-13. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen integrin avP3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen integrin a5pi. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen KIR. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen LAG-3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen Lewis-Y antigen. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen LY6K. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MAGE-1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MAGE- A3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MAGE-C2. In some embodiments, the extracellular antigen binding domain binds the tumorATTORNEY DOCKET NO. MIL-052WO1associated antigen MAGE-D4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MAPG. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MART-1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen Melan-A. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MET. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen mesothelin. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MIF. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MSLN (Mesothelin). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MS4A1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen mucin CanAg. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MUC1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MUC4. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen MUC1 6. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen NG2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen N-glycolylneuraminic acid. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen Notch receptor PD-1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen NY-ESO-1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen OCAA. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PAP. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PDGF-Ra. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PDCD1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PD1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PD-L1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen phosphate-sodium co-transporter. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen phosphatidylserine. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PRAME. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen PSA. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen RANKL. In some embodiments,ATTORNEY DOCKET NO. MIL-052WO1the extracellular antigen binding domain binds the tumor associated antigen RON. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen R0R1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen Sialyl-Tn. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen SLAMF7. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen SPAG-9. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen SSX1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen STEAP1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen surviving. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TAG-72. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen telomerase. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TEM1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen tenascin C. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TGF-p. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TFM-3. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TRAIL-R2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TRAIL-R1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TWEAK receptor. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen tumor specific glycosylation of MUC1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen tumor-associated calcium signal transducer 2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen tumor antigen CTAA16.88. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen TYRP1 (glycoprotein 75). In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen VEGF-A. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen VEGFR2. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen VEGFR-1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen vimentin. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen VISTA. In some embodiments, the extracellular antigen binding domain binds theATTORNEY DOCKET NO. MIL-052WO1tumor associated antigen WT1. In some embodiments, the extracellular antigen binding domain binds the tumor associated antigen XAGE-lb.

[0019] In some embodiments, the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) binding domain that binds GUCY2C.

[0020] In some embodiments the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises: a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of HYYWS (HCDR1; SEQ ID NO: 23), RIYPSGSTSYNPSLKS (HCDR2; SEQ ID NO: 26), and DRSTGWSEWNSDL (HCDR3; SEQ ID NO: 31); a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMS (HCDR1; SEQ ID NO: 24), KIRHDGGEKYYVDSVKG (HCDR2; SEQ ID NO: 27), and DYTRDV (HCDR3; SEQ ID NO: 32); a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIKYDGSEKYYADSVKG (HCDR2; SEQ ID NO: 28), and DYNKDY (HCDR3; SEQ ID NO: 33); a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIRHDGGEKYYPDSVKG (HCDR2; SEQ ID NO: 29), and DYNKDL (HCDR3; SEQ ID NO: 34) or a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIRHDGGEKYYADSVKG (HCDR2; SEQ ID NO: 28), and DYNKDY (HCDR3; SEQ ID NO: 33). In some embodiments the extracellular antigen binding domain is an antiguanylate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises the heavy chain variable region (VH) with complementarity determining region (CDR) sequences of HYYWS (HCDR1; SEQ ID NO: 23), RIYPSGSTSYNPSLKS (HCDR2; SEQ ID NO: 26), and DRSTGWSEWNSDL (HCDR3; SEQ ID NO: 31). In some embodiments the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises the heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMS (HCDR1), KIRHDGGEKYYVDSVKG (HCDR2; SEQ ID NO: 27), and DYTRDV (HCDR3; SEQ ID NO: 32).). In some embodiments the extracellular antigen binding domain is an antiguanylate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises the heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIKYDGSEKYYADSVKG (HCDR2; SEQ ID NO: 28), and DYNKDY (HCDR3; SEQ ID NO: 33). In some embodiments the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) bindingATTORNEY DOCKET NO. MIL-052WO1domain that binds GUCY2C and comprises the heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 25), KIRHDGGEKYYPDSVKG (HCDR2; SEQ ID NO: 29), and DYNKDL (HCDR3; SEQ ID NO: 34). In some embodiments the extracellular antigen binding domain is an antiguanylate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises the heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIRHDGGEKYYADSVKG (HCDR2; SEQ ID NO: 28), and DYNKDY (HCDR3; SEQ ID NO: 33).

[0021] In some embodiments, the CAR comprises a CD28 transmembrane domain. In some embodiments, the CAR comprises a CD8 transmembrane domain. In some embodiments, the CAR comprises one or more costimulatory domain. In some embodiments, the CAR comprises a costimulatory domain derived from 4- IBB. In some embodiments, the CAR comprises a costimulatory domain derived from CD28.

[0022] In some embodiments, the immune cell further expresses an fL-2RaPy.

[0023] In some embodiments, the immune cell further expresses a recombinant IL-2Rp.

[0024] In some embodiments, the immune cell comprises a switch receptor comprising Fas and / or 0X40. In some embodiments, the immune cell comprises a switch receptor comprising Fas. In some embodiments, the immune cell comprises a switch receptor comprising 0X40. In some embodiments, the immune cell comprises a switch receptor comprising Fas and 0X40.

[0025] In some embodiments, the immune cell further expresses a polypeptide comprising a dominant-negative Fas (Fas-DN), a Fas-CD27 chimeric polypeptide (Fas-CD27), a Fas-4-1BB chimeric polypeptide (Fas-BB), a Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof. In some embodiments, the immune cell further expresses a polypeptide comprising a dominant-negative Fas (Fas-DN). In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-CD27 chimeric polypeptide (Fas-CD27). In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-4-lBB chimeric polypeptide (Fas-BB). In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-OX40 chimeric polypeptide (Fas-OX40). In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-DN and a Fas-CD27. In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-CD27 and a Fas-BB. In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-BB and a Fas-OX40. In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-OX40 and a Fas-DN. In someATTORNEY DOCKET NO. MIL-052WO1embodiments, the immune cell further expresses a polypeptide comprising a Fas-DN and a Fas-BB. In some embodiments, the immune cell further expresses a polypeptide comprising a Fas-CD27 and a Fas-OX40. In some embodiments, the immune cell further expresses a Fas-BB and a Fas-DN. In some embodiments, the immune cell further expresses a polypeptide comprising any combination of three or more of the foregoing.

[0026] In some embodiments, NKp30 and CD56 expression levels are elevated.

[0027] In some embodiments, the immune cell comprises greater cytotoxicity, potency, proliferation and / or anti-tumor activity relative to a control immune cell that does not express an IL-2 mutein.

[0028] In some embodiments, the IL-2 mutein is expressed under an inducible promoter.

[0029] In some embodiments, the inducible promoter is an NF AT minimal promoter.

[0030] In some embodiments, the IL-2 mutein is expressed under a constitutive promoter.

[0031] In some embodiments, the constitutive promoter is 5' LTR or EFS in SIN vector.

[0032] In some embodiments, the immune cell does not express a recombinant IL- 13 superkine.

[0033] In some embodiments, the immune cell is a cord-blood derived natural killer (CB-NK) cell.

[0034] In some embodiments, the immune cell is a y5 T cell.

[0035] In some embodiments, the y5 T cell is from blood.

[0036] In some embodiments, the y5 T cell is isolated from the skin.

[0037] In some embodiments, the y5 T cell is isolated / derived from a tissue (e.g., either a hematopoietic or non-hematopoietic tissue sample) or derived from a pluripotent stem cell.

[0038] In some embodiments, the y5 T cell is a V51 (Vdeltal) cell.

[0039] In some embodiments, the immune cell is allogeneic.

[0040] In some embodiments, a polynucleotide encoding the IL-2 mutein described herein is provided.

[0041] In some embodiments, a method of modulating immune function comprises administering to a patient in need thereof an immune cell of the previous embodiments.

[0042] In some embodiments, administration of the immune cell leads to increased proliferation of immune cells, cytotoxicity, potency and / or tumor killing. In some embodiments, administration of the immune cell leads to increased proliferation of immune cells, In some embodiments, administration of the immune cell leads to increased cytotoxicity. In some embodiments, administration of the immune cell leads to increased potency. In some embodiments, administration of the immune cell leads to increased tumorATTORNEY DOCKET NO. MIL-052WO1killing. In some embodiments, administration of the immune cell leads to increased proliferation of immune cells, cytotoxicity, potency, and tumor killing.

[0043] In some embodiments, the method of treating cancer comprises administering to a patient in need thereof a therapeutically effective amount of the immune cell of the previous embodiments.

[0044] In some embodiments, the cancer is selected from acute myeloid leukemia, gastrointestinal cancer, colorectal cancer, colorectal adenocarcinoma, colorectal leiomyosarcoma, colorectal lymphoma, colorectal melanoma, a colorectal neuroendocrine tumor, metastatic colon cancer, stomach cancer, gastric adenocarcinoma, gastric lymphoma, gastric sarcoma, esophageal cancer, squamous cell carcinoma, adenocarcinoma of the esophagus, and pancreatic cancer. In some embodiments, the cancer is acute myeloid leukemia. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the cancer is colorectal leiomyosarcoma. In some embodiments, the cancer is colorectal lymphoma. In some embodiments, the cancer is colorectal melanoma. In some embodiments, the cancer is a colorectal neuroendocrine tumor. In some embodiments, the cancer is metastatic colon cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is gastric lymphoma. In some embodiments, the cancer is gastric sarcoma. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is adenocarcinoma of the esophagus. In some embodiments, the cancer is pancreatic cancer.BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1 is a schematic representation of cis-activation of an immune effector cell, e.g., an immune cell expressing a chimeric antigen receptor (CAR) or a TCR to minimize systematic exposure. As a non-limiting example, the cis activation comprises a membrane bound attenuated IL-2 with reduced binding affinity to an IL-2 receptor (e.g., IL-2RP).

[0046] FIG. 2A is a graph that shows reduction in tumor cell numbers in the absence of external cytokine support; CB-NK engineered with H9 demonstrated better tumor control in MV-4-11 at E: T ratio of 3: 1. FIG. 2B is a graph that shows reduction in tumor cell numbers in the absence of external cytokine support; CB-NK engineered with H9 demonstrated better tumor control in MOLM-13 at E: T ratio of 3: 1. FIG. 2C is a graph that shows NK-cellATTORNEY DOCKET NO. MIL-052WO1proliferation; CB-NK engineered with H9 demonstrated higher cell numbers on a -er 2-weeks’ period co-culture with MV-4-11 at E: T ratio of 3: 1. FIG. 2D is a graph that shows NK-cell proliferation; CB-NK engineered with H9 demonstrated higher cell numbers on a 2-weeks’ period co-culture with MOLM-13 atE: T ratio of 3:1. FIG. 2E is a histogram showing reduction in the number of tumor cells on DI 1, in CB-NK cells engineered with sH9, sIL2, and sIL15; more tumor cells were detected in no cytokine (UTD), or CD123-CAR-sIL15 groups as measured by flow cytometry. FIG. 2F is a histogram that shows cell numbers detected by flow cytometry in CB-NK cells engineered with sH9, compared to sIL15, were greater than CD123-CAR-sIL15. FIG. 2G shows CK analysis by ddPCR demonstrated comparable cell survival or proliferation in CB-NK cells engineered with sH9 and IL-15.

[0047] FIG. 3A is a histogram that shows fold expansion of blood derived V51 T cells from D5 to D14. FIG. 3B is a histogram that shows transduction efficiency of the anti-mesothelin CAR. FIG. 3C is a histogram that shows gMFI of anti-mesothelin CAR. FIG. 3D shows results of IncuCyte imaging-based assay to evaluate H9+IL2RP function, in vitro Repeat Antigen Stimulation (RAS) assay. FIG. 3E is a histogram that shows results of flow cytometry-based RAS assay. FIG. 3F is a histogram that shows results of flow cytometry performed to monitor V51 proliferation. FIG. 3G shows bioluminescence images taken on several timepoints as indicated. FIG. 3H is a graph that shows tumor control in tested samples on a linear scale. FIG. 31 shows tumor control in tested samples in log format. FIG.3 J shows CAR copy number per microgram of DNA normalized to actin of the indicated engineered V51 T cells on day 7 and day 21. Anti-mesothelin CAR only; anti-mesothelin CAR-mbIL15 / Ra + IL2R|J: anti-mesothelin CAR, membrane-bound IL- 15 and IL2RP coexpression; anti-mesothelin_sH9 + IL2R|J: anti-mesothelin CAR, secreted H9, and IL2RP co-expression; anti-mesothelin_sIL2+ IL2R|J: anti-mesothelin CAR, secreted wild-type IL-2, and IL2RP co-expression; anti-mesothelin_sH9T + IL2R|J: anti-mesothelin CAR, secreted H9T, and IL2RP co-expression.

[0048] FIG. 4A and 4B are representative diagrams of the exemplary chimeric antigen receptor (CAR) constructs showing exemplary domains with the CAR.

[0049] FIG. 5A depicts the fold expansion of cells co-expressing exemplary IL-2 muteins and attenuated IL-2 muteins with GCC CAR with or without IL-2Rp. FIG. 5B shows a bar chart of the percent CAR expression when co-expressed with exemplary membrane-bound IL-2 attenuated muteins. FIG. 5C shows RAS assays of cells challenged with HT-29 / GCC of exemplary attenuated muteins co-expressed with CARs versus membrane-bound IL- 15 and H9 IL-2Rp. FIG. 5D depicts a plot of V51 cell proliferation upon repeated antigenATTORNEY DOCKET NO. MIL-052WO1stimulation with HT29 / GCC with attenuated IL-2 muteins. FIG. 5E shows a bar graph of the STAT5 pathway activation in STAT5 reporter cell lines co-cultured with of CAR- y5T cells expressed with exemplary membrane-bound IL-2 muteins. The higher STAT5 signal suggests stronger trans-presentation activity of the exemplary membrane-bound IL-2 muteins.

[0050] FIGs. 6A-6B shows bar graphs of the fold expansion (FIG. 6A) and GCC CAR expression (FIG. 6B) with exemplary attenuated IL-2 muteins in comparison to untransduced cells and membrane-bound IL-15. FIG. 6C is a bar graph of the area under curve (AUC) for all GCC expressing constructs upon repeated antigen stimulation (RAS) with HT29-GCC from FIG. 5C. FIG. 6D shows a plot of V51 cell proliferation upon repeated antigen stimulation with HT29 / GCC.

[0051] FIGs. 7A-7C show the effects of exemplary attenuated IL-2 muteins on signaling, transduction efficiency, expression, in vitro killing and proliferation. FIG. 7A is a bar plot showing that the secreted or membrane bound IL-2 (sIL2 or mbIL2) muteins have attenuated JAK / STAT5 signaling relative to wild-type IL-2 in a reporter cell line. FIG. 7B depicts mbIL2 armoring expression (top) and IL-2 trans-presentation in STAT5 reporter assay (bottom), The results indicate low affinity mbIL2-AE88R exhibits high armoring expression while maintaining low levels of trans-presentation. FIG. 7C shows mb IL-2 activity; the results indicate low affinity mbIL2-AE88R has potent activity in RAS assay. FIG. 7D is a bar chart depicting the signaling of IL-2Ra biased mbIL2 muteins versus non-IL-2Ra-biased mbIL2 muteins on signaling in a reporter cell line. FIG. 7E shows the transduction efficiency of HEK-Blue IL-2 / IL-15 cells transduced with CAR or armoring molecules.

[0052] FIGs. 8A-8H demonstrate that the surface expression of exemplary attenuated IL-2 muteins can be regulated by using different signal peptide sequences across two donors. FIGs. 8A-8B show the expression of the IL-2 mutein as a function of comprising a long signal peptide (LSP). FIGs. 8C-8D describe the gMFI from flow cytometry for IL-2 and GCC expression, respectively. FIGs. 8E-8H show the results of repeated antigen stimulation assays and proliferation of V51 cells using CAR-T cells armored with the exemplary IL-2 muteins across two donors with and without the LSP.

[0053] FIG. 9 shows that exemplary GCC armored CARs expressing mbIL2AE88R exhibit single agent activity with gain-of-activity of armoring versus unarmored GCC CARs in effecting anti-tumor efficacy in mice.

[0054] FIGs. 10A-10G describe the killing capacity of exemplary anti-CD19 CAR-yS T cells expressing IL-2Ra biased mbIL2 muteins versus non-IL-2Ra-biased mbIL2 muteins on tumor killing, y5 T cell proliferation, and CAR maintenance. FIG. 10A is a bar plot showingATTORNEY DOCKET NO. MIL-052WO1the area under curve (AUC) analysis of tumor killing by armored CD 19 CAR-T cells expressing IL-2Ra biased mbIL2 muteins versus non-IL-2Ra-biased mbIL2 muteins (raw data shown in FIGs. 10B-10C for non-biased and biased IL-2 muteins, respectively). FIG.10D-10E describe the y5 T cell proliferation of non-IL-2Ra-biased and IL-2Ra-biased IL-2 muteins, respectively. FIGs. 10F-10G depict the maintenance of the CAR for cells expressing non-IL-2Ra-biased and IL-2Ra-biased IL-2 muteins, respectively.DETAILED DESCRIPTIONDefinitions

[0055] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0056] Administer'. As used herein, “administering” a composition to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

[0057] Affinity: As used herein, the term “affinity” refers to the characteristics of a binding interaction between a binding moiety (e.g., an antigen binding agent (e.g., variable domain described herein) and a target (e.g., an antigen (e.g., GUCY2C)) and that indicates the strength of the binding interaction. In some embodiments, the measure of affinity is expressed as a dissociation constant (KD). In some embodiments, a binding moiety has a high affinity for a target (e.g., a KD of less than about 10⁻⁷ M, less than about 10⁻⁸ M, or less than about 10⁻⁹ M). In some embodiments, a binding moiety has a low affinity for a target (e.g., a KD of higher than about 10⁻⁷M, higher than about 10⁻⁶ M, higher than about 10⁻⁵ M, or higher than about 10⁻⁴M).

[0058] Antibody or Antigen Binding Agent. As used herein, the term “antibody” or “antigen binding agent” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. Those skilled in the art will appreciate that the terms may be used herein interchangeably. In some embodiments, as used herein, the term “antibody” or “antigen binding agent” also refers to an “antibody fragment” or “antibody fragments” or “antigen binding portion”, which includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of “antibody fragments” include Fab, Fab’, F(ab’)2, and FvATTORNEY DOCKET NO. MIL-052WO1fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and CDR-containing moieties included in multi-specific antibodies formed from antibody fragments. Those skilled in the art will appreciate that the term “antibody fragment” does not imply and is not restricted to any particular mode of generation. An antibody fragment may be produced through use of any appropriate methodology, including but not limited to cleavage of an intact antibody, chemical synthesis, recombinant production, etc. As is known in the art, intact antibodies as produced in nature are approximately 150 kDa tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kDa each) and two identical light chain polypeptides (about 25 kDa each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)-an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy -terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy -terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another, and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two P sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel P barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the P sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. Amino acid sequence comparisons among antibody polypeptide chains have defined two light chain (K and X) classes, several heavy chain (e.g., p, y, a, a, 5) classes, and certain heavy chain subclasses (al, a2, yl, y2, y3, and y4). Antibody classes (IgA (e.g., IgAl, IgA2), IgD, IgE,ATTORNEY DOCKET NO. MIL-052WO1IgG (e.g., IgGl, IgG2, IgG3, and IgG4), and IgM are defined based on the class of the utilized heavy chain sequences.

[0059] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In some embodiments, the term refers to a range of values that fall within 10% of the stated reference value. In some embodiments, the term refers to a range of values that fall within 5% of the stated reference value. The term “between” includes the values of the specified boundaries and all intervening values and fractions.

[0060] Autologous'. As used herein, the term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced.

[0061] Allogenic. As used herein, the term “allogenic” refers to material derived from one individual administered to a different individual or individuals.

[0062] Chimeric Antigen Receptor (CAR) '. As used herein, the term “chimeric antigen receptor” or “CAR” refers to a protein that when expressed on the surface of a cell allows a CAR expressing cell to recognize its specific protein (antigen), such as on tumor cells, infected cells or cells mediating autoimmune or inflammatory diseases or disorders. Such receptors are also known as chimeric T cell receptors, chimeric immunoreceptors, or artificial T cell receptors (for example, when expressed on T cells). Upon transduction of a cell with a nucleic acid construct encoding a CAR, the cell will recognize the antigen specified by the CAR. A CAR is typically comprised of an ectodomain (extracellular domain) and an endodomain (cytoplasmic or intracellular domain), separated by a transmembrane domain. The ectodomain, expressed on the surface of the cell, comprises an antigen binding domain or receptor domain, optionally a signal peptide that directs the antigen binding domain into the endoplasmic reticulum for processing, and optionally a spacer (or hinge) region. The antigen binding domain (or receptor domain) comprises peptides that specifically recognize a target antigen. As a non-limiting example, the antigen binding domain can be a single chain antibody, such as an scFv. The spacer region links the antigen binding domain to the transmembrane domain and is designed to be sufficiently flexible to allow the antigen binding domain to orient in a manner that allows antigen recognition. Examples of spacerATTORNEY DOCKET NO. MIL-052WO1domains include, but are not limited to, the hinge region from IgG, the CH2CH3 region of an immunoglobulin, CD28 hinge, Dap 10 hinge, CD8 hinge, and portions of CD3 molecules. The transmembrane domain is a hydrophobic a helix, typically, that spans across the lipid bilayer of the cell membrane. The endodomain of the CAR is composed of a signal transmitting peptide that transmits an activation signal intracellularly to the cell cytoplasm, thereby stimulating the cell expressing the CAR. The endodomain may include multiple such signaling domains, as explained, infra. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some embodiments, the set of polypeptides encoding the CAR are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., an scFv) during cellular processing and localization of the CAR to the cellular membrane. In some embodiments, a CAR is co-ATTORNEY DOCKET NO. MIL-052WO1expressed with IL-2, IL-2 mutein, or membrane-bound variant thereof, to promote CAR-T cell survival.

[0063] Complementarity Determining Region (CDR)'. A “CDR” of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and / or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D. C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g, Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include the “AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the “contact definition” of CDRs based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g, Makabe et al., Journal of Biological Chemistry, 283: 1 156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. Unless stated otherwise, as used herein, CDR definitions are according to Kabat CDRs.

[0064] Comprise: As used herein, the term “comprises” and “comprising” and variations thereof (e.g., “comprises / comprising”, “includes / including”) should be understood to imply the inclusion of a stated component, feature, element or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element or step or group of components, features, elements or steps. Any one of the terms “comprising”, “consisting essentially of’, and “consisting of’ may be substituted with either of the other two terms, while retaining their ordinary meaning.

[0065] Engineered protein: As used herein, “Engineered IL-2,” or “Engineered protein”, or “IL-2 mutein” encompasses an IL-2 having at least one residue that differs from a native or wild-type IL-2, and includes full-length IL-2, truncated forms of IL-2, and forms where IL-2 is linked or fused with another molecule, such as another polypeptide. In some embodiments,ATTORNEY DOCKET NO. MIL-052WO1engineered IL-2 are characterized in having at least one amino acid substitution affecting the interaction of IL-2 with IL-2Rb and / or IL-2Ra. Identification of various engineered forms of IL-2 as described herein are made with respect to the wild-type IL-2 sequence. As used herein, “IL-2” and “IL2” are used interchangeably.

[0066] Epitope: As used herein, the term “epitope” includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component in whole or in part. In some embodiments, an epitope is comprised of a plurality of amino acids in an antigen. In some embodiments, such amino acid residues are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, the amino acid residues are physically near to or contour with each other in space when the antigen adopts such a conformation. In some embodiments, at least some of the amino acids are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized; e.g., a non-linear epitope).

[0067] Immune Response'. As used herein, the term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection).Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of immune cells (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like.

[0068] In vitro'. As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0069] In vivo'. As used herein, the term “in vivo" refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro or ex vivo systems).

[0070] Isolated. As used herein, the term "isolated" substance, such as an isolated nucleic acid, is a substance that is not in its natural environment, although the isolated substance need not be purified. For example, an isolated nucleic acid is a nucleic acid that is not produced orATTORNEY DOCKET NO. MIL-052WO1located in its natural or native environment (e.g., a cell). The isolated material may be isolated, fractionated, or at least partially purified by any suitable technique.

[0071] Mutein'. As used herein, the term “mutein” or “IL-2 mutein” is used as a synonym for Engineered protein, wherein the term encompasses a mutated protein (henceforth “mutein”) that differs by one amino acid than the wild-type protein, is truncated, and the like.

[0072] Polypeptide'. A “polypeptide”, generally speaking, is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally. In some embodiments, the term “polypeptide” is used to refer to specific functional classes of polypeptides, such as, an antibody, chimeric antigen receptor, or costimulatory domain polypeptides, etc. For each such class, the present specification provides and / or the art is aware of several examples of amino acid sequences of known exemplary polypeptides within the class; in some embodiments, one or more such known polypeptides is / are reference polypeptides for the class. In such embodiments, the term “polypeptide” refers to any member of the class that shows sufficient sequence homology or identity with a relevant reference polypeptide that one skilled in the art would appreciate that it should be included in the class. In many embodiments, a member of the representative class also shares significant activity with the reference polypeptide. For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (z.e., a conserved region, often including a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

[0073] It is understood that the antibodies and antigen binding agents of the invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity, can be determined as described in Bowie, J U et al. Science 247:1306-1310 (1990)ATTORNEY DOCKET NO. MIL-052WO1or Padlan et al. FASEB J. 9:133-139 (1995). A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), P-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0074] Single domain antibody, as used herein, the terms “single domain antibody (sdAb)”, “variable single domain” or “immunoglobulin single variable domain (ISV)” “single heavy chain variable domain (VH) antibody” refer to the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. A sdAb is a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. A VH single domain antibody refers to a single domain antibody that has a human heavy chain variable domain or a domain that is derived from a human heavy chain variable domain. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred to as “VHHs”. Some VHHs may also be known as Nanobodies. Camelid sdAb is one of the smallest known antigen-binding antibody fragments. See, e.g, Hamers-Casterman et al., Nature 363: 446-8 (1993); Greenberg et al., Nature 374: 168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-26 (2013). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0075] Subject or Patient: As used herein, “subject” or “patient” refers to an individual suffering from acute myeloid leukemia, wherein the acute myeloid leukemia is relapsed or refractory.

[0076] Therapeutic agent: As used herein, the term “therapeutic agent” refers to an agent (e.g., an antigen binding agent) that has biological activity. The term is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. In some embodiments, the therapeutic agent may be an anti-cancer agent or a chemotherapeutic agent. As used herein, the terms “anti-cancerATTORNEY DOCKET NO. MIL-052WO1agent” or “chemotherapeutic agent” refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis or angiogenesis is frequently a property of anti-cancer or chemotherapeutic agents. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent which inhibits or suppresses cell growth and / or multiplication of cells. In some embodiments, the therapeutic agent is a genetically modified cell or antibody.. In some embodiments, the therapeutic agent is a cell (e.g., a population of cells) expressing an IL-2 mutein as described herein. In some embodiments, the therapeutic agent is a cell expressing an IL-2 mutein and a CAR.

[0077] Therapeutically effective amount. As used herein, the term “therapeutically effective amount” means an amount effective, at dosages, frequency of administration and for duration of time necessary to achieve the desired results such that one or more symptoms or biomarkers is improved after treatment.

[0078] Treat or Treatment. As used herein, the term “treat” or “treatment” is defined as the administration of cells comprising an IL-2 mutein to a subject, e.g., a patient, or administration, e.g., by application, to an isolated tissue or cell from a subject which is returned to the subject. The IL-2 mutein expressing cell can be administered alone or in combination with a second agent. The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder, e.g., a cancer. While not wishing to be bound by theory, treating is believed to cause the inhibition, ablation, or killing of a cell in vitro or in vivo, or otherwise reducing capacity of a cell, e.g., an aberrant cell, to mediate a disorder, e.g., a disorder as described herein (e.g., a cancer).

[0079] Variable region or domain'. As used herein, the terms “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies have a single heavy chain variable region.

[0080] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNAATTORNEY DOCKET NO. MIL-052WO1segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0081] Wild-type: As used herein, “Wild-type” or “native” when used in reference to IL-2 is intended to mean the mature human IL-2 molecule, for example, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 12).Interleukin 2 (IL-2) and IL-2 Receptor

[0082] Interleukin 2 (IL-2) is a cytokine that regulates immune cell proliferation and activation. It has a length of 133 amino acids and the structure comprises four antiparallel amphipathic C-helices. As used herein, IL-2 and IL2 are used interchangeably. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R) which comprises up to three separate subunits. The association of all three subunits, the interleukin-2 receptor a chain (IL-2R a, or CD25), the interleukin-2 receptor P chain (IL-2R P, or CD 122), and the interleukin-2 receptor gamma chain (IL-2R y, or CD 132), produces trimeric IL-2R aPy, which is a high affinity receptor for IL-2. The association of IL-2RP with the IL-2Ry subunit produces the dimeric receptor IL-2RPy and is referred to as intermediate affinity IL-2R. The IL-2R a subunit forms a monomeric low affinity IL-2 receptor. Expression of IL-2Ra is involved in the expansion of immunosuppressive regulatory T cells (Tregs); while dimeric IL-2RPy in the absence of IL-2Ra leads to cytolytic CD8+ T cells and NK cells proliferate and kill. IL-2 signals via three principal signaling pathways, the JAK-STAT pathway (mainly activating JAK1, JAK3, STAT5A, and STAT5B), the RAS-MAP kinase pathways, and the PI 3-kinase-AKT pathway, which together contribute to the range of biological actions mediated by IL-2. However, systemic administration of IL-2 often leads to severe toxicity. To address this, immune cells are engineered to express an IL-2 receptor p (IL-2RP) that can only be activated by a modified, orthogonal IL-2. Orthogonal IL-2RP was introduced to immune cells targeting CD 19, and the immune cells were transferred to mice bearing leukemias or lymphomas. Administration of orthogonal IL-2 to immune cell recipients drove activation, expansion, and antitumor efficacy of immune cells in both preclinical models with limited systemic toxicity.ATTORNEY DOCKET NO. MIL-052WO1

[0083] In some embodiments, high-dose IL-2 induces a selective expansion of regulatory T (Treg) cells, which limits the activity of NK cells, resulting in poor clinical responses to IL-2 therapy. In some embodiments, the depletion of Treg cells leads to increased IL-2 availability for NK cells to increase IFN-y production and cytotoxicity. To overcome Treg cell inhibition, a mutant form of IL-2 or IL-2 variant that preferentially binds to CD122 / CD132 and that has reduced binding to CD25 is used. In some embodiments, the IL-2 mutein comprises one or more mutations at positions 18, 22, 38, 42, 80, 81, 85, 86, 88, 91, 92, and 126 of SEQ ID NO: 12. In some embodiments, the IL-2 mutein comprises one or more mutations selected from L18R, Q22E, R38E, F42A, L80F, R81D, L85V, I86V, N88R, N88T, V91T, I92F, Q126A, Q126C, Q126E, Q126H, Q126K, and Q126T.

[0084] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions L80, R81, L85, 186, and 192 according to SEQ ID NO: 12.

[0085] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions L80, R81, L85, 186, 192 and Q126 according to SEQ ID NO: 12.

[0086] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions L80, R81, L85, 186, 192, L18, Q22, and Q126 according to SEQ ID NO: 12.

[0087] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions F42 and R38 according to SEQ ID NO: 12.

[0088] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions F42, R38 and V91 according to SEQ ID NO: 12.

[0089] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions F42, R38 and Q126 according to SEQ ID NO: 12.

[0090] In some embodiments, the IL-2 mutein comprises amino acid substitutions at positions F42, R38 and N88 according to SEQ ID NO: 12.

[0091] Exemplary IL-2 muteins comprise any one of SEQ ID NO: 1-11.

[0092] H9 IL-2 Mutein (L80F, R81D, L85V, I86V, I92F) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 1, H9)

[0093] In some embodiments, IL-2 muteins comprising SEQ ID NO: 1 are also referred to in this application as H9.

[0094] H9T IL-2 Mutein (L80F, R81D, L85V, I86V, I92F, Q126T)ATTORNEY DOCKET NO. MIL-052WO1APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CTSI ISTLT (SEQ ID NO: 2, H9T)

[0095] In some embodiments, IL-2 muteins comprising SEQ ID NO: 2 are also referred to in this application as H9T.

[0096] H9REH IL-2 Mutein (L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126H) APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CHSI ISTLT (SEQ ID NO: 3, H9REH)

[0097] In some embodiments, IL-2 muteins comprising SEQ ID NO: 3 are also referred to in this application as H9REH.

[0098] H9REK IL-2 Mutein (L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126K) APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CKSI ISTLT (SEQ ID NO: 4, H9REK)

[0099] In some embodiments, IL-2 muteins comprising SEQ ID NO: 4 are also referred to in this application as H9REK.

[0100] H9REA IL-2 Mutein (L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126A) APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CASI ISTLT (SEQ ID NO: 5, H9REA)

[0101] In some embodiments, IL-2 muteins comprising SEQ ID NO: 5 are also referred to in this application as H9REA.

[0102] H9REC IL-2 Mutein (L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126C) APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITF CCSI ISTLT (SEQ ID NO: 6, H9REC)

[0103] In some embodiments, IL-2 muteins comprising SEQ ID NO: 6 are also referred to in this application as H9REC.

[0104] IL2AE IL-2 Mutein (F42A, R38E) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 7, IL2AE)ATTORNEY DOCKET NO. MIL-052WO1

[0105] In some embodiments, IL-2 muteins comprising SEQ ID NO: 7 are also referred to in this application as IL2AE.

[0106] IL2AET IL-2 Mutein (F42A, R38E, V91T) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVITLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 8, IL2AET)

[0107] In some embodiments, IL-2 muteins comprising SEQ ID NO: 8 are also referred to in this application as IL2AET.

[0108] IL2AEE IL-2 Mutein (F42A, R38E, Q126E) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CESI ISTLT (SEQ ID NO: 9, IL2AEE)

[0109] In some embodiments, IL-2 muteins comprising SEQ ID NO: 9 are also referred to in this application as IL2AEE.

[0110] IL2AE88T IL-2 Mutein (F42A, R38E, N88T) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNITVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 10, IL2AE88T)

[0111] In some embodiments, IL-2 muteins comprising SEQ ID NO: 10 are also referred to in this application as IL2AE88T.

[0112] IL2AE88R IL-2 Mutein (F42A, R38E, N88R) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEE LKPLEEVLNLAQSKNFHLRPRDLISNIRVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSI ISTLT (SEQ ID NO: 11, IL2AE88R)

[0113] In some embodiments, IL-2 muteins comprising SEQ ID NO: 11 are also referred to in this application as IL2AE88R.

[0114] In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 70-75% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 75-80% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 80-85% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 85-90% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 90-95% identical toATTORNEY DOCKET NO. MIL-052WO1any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 70% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 72% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 74% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 75% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 78% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 80% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 81% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 82% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 83% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 84% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 85% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 86% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 87% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 88% identical to any one of SEQ ID NO: 11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 89% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 90% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 90% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 90% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 91% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 92% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of IL-2 mutant is 93% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 94% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutant is 95% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 96% identical to any one of SEQ ID NOs: 1-11. In someATTORNEY DOCKET NO. MIL-052WO1embodiments, an exemplary an IL-2 mutein is 97% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 98% identical to any one of SEQ ID NOs: 1-11. In some embodiments, an exemplary amino acid sequence of an IL-2 mutein is 99% identical to any one of SEQ ID NOs: 1-11.

[0115] In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:1. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:2. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:3. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:4. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:5. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:6. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:7. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO:9. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence 70% identical to SEQ ID NO: 11.

[0116] In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO:1. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO:2. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO:9. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence 80% identical to SEQ ID NO: 11.

[0117] In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NON. In some embodiment, the IL-2 muteinATTORNEY DOCKET NO. MIL-052WO1comprises an amino acid sequence 85% identical to SEQ ID NO:4. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO:5. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO:6. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO:7. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO:9. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence 85% identical to SEQ ID NO: 11.

[0118] In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:1. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:2. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:3. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:5. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:6. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:7. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO:9. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence 90% identical to SEQ ID NO: 11.

[0119] In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NO:1. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NO:2. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NO:9. In some embodiment, the IL-2 muteinATTORNEY DOCKET NO. MIL-052WO1comprises an amino acid sequence 95% identical to SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence 95% identical to SEQ ID NO: 11.

[0120] In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:1. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:2. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:3. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NON. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:5. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:6. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:7. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:8. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:9. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO: 10. In some embodiment, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:11.

[0121] In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NOs: 1-11. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 1. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 2. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 3. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 4. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 5. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 6. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 7. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 8. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 9. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 10. In some embodiments, the IL-2 comprises a nucleic acid sequence encoding SEQ ID NO: 11.

[0122] In some embodiments, the IL-2 mutein has reduced binding to IL-2Rp. In some embodiments, the IL-2 mutein has reduced binding to IL-2RPG. In some embodiments, the IL-2 mutein has reduced binding to IL-2Rp. In some embodiments, the IL-2 mutein has reduced binding to IL-2Ra. In some embodiments, the IL-2 mutein further comprises a signal peptide. In some embodiments, the signal peptide is a long signal peptide. In some embodiments, the signal peptide is a native signal peptide of a naturally occurring protein. In some embodiments, the signal peptide is a synthetic peptide or a heterologous peptide. InATTORNEY DOCKET NO. MIL-052WO1some embodiments, a sequence encoding an appropriate native orheterologous signal sequence is added to the nucleic acid sequence encoding an IL-2 mutein. In some embodiments, the signal peptide is 10-100 amino acids in length. In some embodiments, the signal peptide is 10-30, 15-50, 15-30, 40-100 amino acids in length. In some embodiments, the signal peptide is a signal peptide from IL-15.

[0123] In some embodiments, the IL-2 mutein comprises a signal peptide having the sequence of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 74).

[0124] In some embodiments, IL-2 mutein is a soluble form of IL-2.

[0125] In some embodiments, IL-2 muteins enhance affinity at one receptor binding site (IL-2RP) while attenuating interactions at the second receptor binding site (yc) in order to manipulate dimerization and signal initiation. Because of augmented binding to IL-2RP, these molecules were dominant over endogenous IL-2. In some embodiments, the IL-2 muteins bind IL-2RP or IL-2Rycwith high affinity in the absence of IL-2Ra. In some embodiments, the IL-2 muteins block IL-2Ra induction, prolonged survival, and potently inhibited the spontaneous proliferation of peripheral blood T cells.

[0126] In some embodiments, the IL-2 mutein is membrane bound.

[0127] In some embodiments, the membrane bound IL-2 comprises a transmembrane domain. In some embodiments, the membrane bound IL-2 further comprises a hinge domain.Membrane Bound IL-2 Mutein Componentsa. Membrane Bound IL-2 Mutein Hinge Domain

[0128] A membrane bound IL-2 mutein can include the structure of a spacer or hinge domain between the IL-2 mutein and a transmembrane domain. The membrane bound IL-2 mutein hinge domain is a spacer that provides separation of the IL-2 mutein from the cell membrane and an intracellular signaling module. One of ordinary skill in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates flexibility.See, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004). The membrane bound IL-2 mutein hinge sequence can be any suitable sequence derived or obtained from any suitable molecule such as a human protein. In some embodiments, the length of the hinge sequence may be optimized based on the IL-2 mutein.ATTORNEY DOCKET NO. MIL-052WO1

[0129] A membrane bound IL-2 mutein contemplated herein comprises a hinge sequence between the IL-2 mutein and a transmembrane domain. In some embodiments, the membrane bound IL-2 mutein hinge domain comprises a CD4, CD8, CD28, or B7 hinge domain.

[0130] In some embodiments, the membrane bound IL-2 mutein hinge domain comprises a CD8 hinge domain.b. Membrane Bound IL-2 Mutein Transmembrane Domain

[0131] With respect to the transmembrane domain, a membrane bound IL-2 mutein can be designed to comprise a transmembrane domain that is fused to the IL-2 mutein, e.g., through a hinge sequence. In some embodiments, the membrane bound IL-2 mutein transmembrane domain connects an intracellular signaling domain to the hinge region of the IL-2 mutein.

[0132] The membrane bound IL-2 mutein transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the a, P or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 a, CD8 P), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. In some embodiments, the membrane bound IL-2 mutein transmembrane domain comprises a transmembrane domain selected from CD4, CD8, CD28, or B7.

[0133] In some embodiments, the membrane bound IL-2 mutein transmembrane domain comprises a CD8 transmembrane domain.c. Membrane Bound IL-2 Mutein Intracellular Domain

[0134] Generally, intracellular or cytoplasmic domains of receptors mediate signaling cascades that are stimulated by the binding of a ligand to a receptor, such as a T cell receptor (TCR) on an immune cell recognizing an antigenic peptide in complex with a major histocompatibility complex. As it pertains to TCR signaling, upon stimulation, one of the proteins that is trafficked to the cell surface via intracellular vesicles is cytotoxic T lymphocyte antigen 4 (CTLA4).

[0135] CTLA4 is a glycoprotein that is expressed by CD4+and CD8+T cells and serves as an essential negative regulator of T cell-mediated immune responses. Importantly, the cytoplasmic domain of CTLA4 controls its trafficking to the cell surface, phosphorylation of its Y164 leads to stable cell surface expression, and its highly conserved YVKM motif promotes endocytosis (L. S. K. Walker etal (2011), Nat. Rev. Immu.. 11: 852-863,ATTORNEY DOCKET NO. MIL-052WO1incorporated by reference). It has been noted in a recent study that the monomeric, duplex, or triplex addition of CTLA-4 cytoplasmic domains to the C-termini of CARs of CAR-T cells exhibit progressive increase in cytotoxicity, reduction in CAR-mediated trogocytosis, and improved CAR-T survival, leading to greater anti-tumor efficacy, which relates to its endocytic activity (X. Zhou et al, 2023, Nat. Immun., 24: 1499-1510, incorporated by reference).

[0136] In some embodiments, the membrane bound IL-2 mutein comprises an intracellular domain.

[0137] In some embodiments, the membrane bound IL-2 mutein intracellular domain comprises a cytoplasmic tail domain of CTLA4.

[0138] In some embodiments, the CTLA4 cytoplasmic tail comprises an amino acid sequence of at least 90% identical to the amino acid sequence SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 70). In some embodiments, the CTLA4 cytoplasmic tail comprises an amino acid sequence of SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 70).

[0139] In some embodiments, the membrane bound IL-2 mutein comprising a CTLA4 intracellular domain is co-expressed with a CAR. In some embodiments, the expression of a membrane bound IL-2 mutein comprising a CTLA4 intracellular domain does not impact CAR expression.

[0140] In some embodiments, the expression of a membrane bound IL-2 mutein comprising a CTLA4 intracellular domain is modulated based on T cell activation.

[0141] As a non-limiting example, the membrane bound IL-2 mutein comprises a CD8 hinge domain and a CD8 transmembrane domain.Immune Cells

[0142] In accordance with the present invention, an immune cell that expresses an IL-2 mutein described herein can be any immune cells. An immune cell can be but is not limited to a T cell, a B cell, a NK cell, a monocyte, a natural killer T cell (NKT) or a regulatory T cell. In some embodiments, the immune cell is a aP T cell. In some embodiments, the immune cell is a y5 T cell.

[0143] In some embodiments, the immune cell is a NK cell, such as a NK tolerant cell, a NK cytotoxic cell and a NK regulatory cell.ATTORNEY DOCKET NO. MIL-052WO1

[0144] The immune cells can be from sources including peripheral blood, bone marrow, tissues (e.g., liver tissue, epithelial tissue), cord blood and pluripotent stem cells. The immune cells can be from any species.Cord blood derived NK cells

[0145] In some embodiments, the cytokine is engineered to express in a Natural killer(NK) cell. In some embodiments, NK cells are from blood. In some cases, NK cells are derived from cord blood.

[0146] NK cells comprising 10-15% of peripheral blood immune cells, play an important role in immune surveillance due to their innate ability to kill cancer and virally infected cells without prior sensitizationl. NK cells are identified by the surface expression of CD56, NKp30 and absence of the T cell marker CD3. A subset of NK cells expresses the FcyRIII protein, CD 16, that enhances NK cell cytotoxic function by aiding in antibody-dependent cellular cytotoxicity (ADCC). NK cell function is largely controlled by families of cell surface activating and inhibitory receptors. Cytokine-based expansion methods result in highly cytotoxic NK cells with memory-like features, but limited fold expansions (~4-fold at day 10 of expansion) have been reported due to NK cell senescence.

[0147] Umbilical cord blood (UCB) is a rich source of NK cells (CB-NK). Additionally, CB-NK cells are easier to stimulate and they have more stable number from donor to donor. UCB-derived NK cells are also younger and have a stronger proliferation potential than the peripheral blood counterparts. In some embodiments, NK cells are isolated from a cryopreserved umbilical cord blood unit. In some embodiments, NK cells can be modified to express a chimeric antigen receptor. As a non-limiting example, NK cells can be modified to express the anti-GCC targeting chimeric antigen receptor (CAR), resulting in GCC-CAR NK cells. As a non-limiting example, NK cells can be modified to express the anti-CD19 targeting chimeric antigen receptor (CAR), resulting in CD19-CARNK cells. In other embodiments, NK cells can be modified to express a T cell receptor (TCR).

[0148] In some embodiments, the cells are human cord blood-derived NK cells.

[0149] In some embodiments, the cells are mouse cord blood-derived NK cells.

[0150] In some embodiments, the cells are rat cord blood-derived NK cells.

[0151] In some embodiments, the present invention provides a NK cell comprising a CAR described herein. In some embodiments, the NK cell may comprise one or more cytokines and / or cytokine receptors. In some embodiments, the NK cell may comprise one or more cytokines and / or cytokine receptors in combination with a CAR.ATTORNEY DOCKET NO. MIL-052WO1

[0152] In some embodiments, the present invention provides a NK cell comprising a polynucleotide encoding a CAR described herein. In some embodiments, the NK cell may comprise a polynucleotide encoding one or more cytokines and / or cytokine receptors. In some embodiments, the NK cell may comprise a polynucleotide encoding one or more cytokines and / or cytokine receptors in combination with a CAR.

[0153] In some embodiments, the present invention provides a NK cell comprising a CAR described herein. In some embodiments, the NK cell comprises a cytokine or cytokine receptor selected from IL-2, and IL-2Ra / IL-2Rp / IL-2Rγ. In some embodiments, the NK cell may comprise a cytokine or cytokine receptor selected from IL-2 and IL-2RP, in combination with a CAR.

[0154] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, and I92F according to wild type IL-2.

[0155] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, and Q126T according to wild type IL-2.

[0156] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126H according to wild-type IL-2.

[0157] In some embodiments, the NK comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126K according to wild type IL-2.

[0158] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126A according to wild type IL-2.

[0159] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126C according to wild type IL-2.

[0160] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, and R38E according to wild type IL-2.

[0161] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and V91T according to wild type IL-2.

[0162] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and Q126E according to wild type IL-2.ATTORNEY DOCKET NO. MIL-052WO1

[0163] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and N88T according to wild type IL-2.

[0164] In some embodiments, the NK cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations F42A, R38E, and N88R according to wild type IL-2.

[0165] In some embodiments, the NK cell may comprise one or more CARs and an IL-2 mutein alone or in combination with IL-2Rp. In some embodiments, the present invention encompasses a V51+ T cell expressing an IL-2 mutein comprising any one of SEQ ID NOs: 1-11. In some embodiments, the present invention encompasses aNK cell expressing IL-2 comprising an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-11.

[0166] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO:1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO:1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO:1. In some embodiments, theNK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO:1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO:1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 1. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO:1.

[0167] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO:2. In some embodiments, the NK cell expressing IL-2ATTORNEY DOCKET NO. MIL-052WO1comprises an amino acid sequence which is 96% identical to SEQ ID NO: 2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 2. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 2.

[0168] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 3.

[0169] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 4. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 4. In someATTORNEY DOCKET NO. MIL-052WO1embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 4.

[0170] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 5. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 5.

[0171] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 6. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 6.

[0172] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2ATTORNEY DOCKET NO. MIL-052WO1comprises an amino acid sequence which is 91% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 7. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 7.

[0173] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 8. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 8.

[0174] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 9. In some embodiments, theATTORNEY DOCKET NO. MIL-052WO1NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 9. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 9.

[0175] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 3. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 10. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 10.

[0176] In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 90% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 91% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 92% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 93% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 94% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 95% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 96% identical to SEQ ID NO:ATTORNEY DOCKET NO. MIL-052WO111. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 97% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 98% identical to SEQ ID NO: 11. In some embodiments, the NK cell expressing IL-2 comprises an amino acid sequence which is 99% identical to SEQ ID NO: 11.

[0177] In some embodiments, the NK comprises a wild-type human IL-2. In some embodiments, the NK cell has reduced binding to IL-2Rp.

[0178] In some embodiments, the NK cell is engineered to express a CAR and a membrane 11-2 mutein. In some embodiments, the NK cell is engineered to express a TCR and a membrane 11-2 mutein. In some embodiments, the NK cell is engineered to express a CAR and a membrane 11-2 mutein and IL-2Rp. In some embodiments, the NK cell is engineered to express a TCR and a membrane 11-2 mutein and IL-2Rp.

[0179] In some embodiments, the NK cell may comprise polynucleotides encoding one or more CARs and an IL-2 mutein alone or in combination with IL-2Rp. In some embodiments, the NK comprising a polynucleotide encoding a wild-type human IL-2. In some embodiments, the NK cell may also comprise a polynucleotide encoding IL-2Rp.Gamma Delta T cells

[0180] In some embodiments, the immune cells are T cells. In some examples, the T cells are aP T cells. In some embodiments, the immune cells are y5 T cells.

[0181] y5 T cells have both adaptive and innate characteristics. These cells have the potential to develop immunological memory. At the same time, they rapidly recognize and respond to ubiquitous changes, but release less cytokines for proliferation. The persistence of such cells in large numbers in vivo is often limited to only a few days. In certain embodiments, the engineered y5 T-cell is selected from the group consisting of: a y952T cell, a 51T cell, a 53T cell, or a combination thereof.

[0182] In some embodiments, a T-cell expressing a cytokine described herein is a y5 T cell.

[0183] A y T cell expresses a yd TCR composed of one gamma chain (y) and one delta (5) chain. The y5 TCR is required for y5 T cell function and development. y5 T cells are non-conventional T cells which show several properties of innate immune cells. y5 T cells present in blood and tissues with a restricted TCR repertoire and circulate as cells with a preactivated phenotype thus being able to generate rapid immune responses. In contrast to aP T cells, y5 T cells exert a direct cytotoxicity and do not require stimulation through CD3ζ to initiate target cell killing. Another advantage of y5 T cells is that ex vivo expanded y5 T cellsATTORNEY DOCKET NO. MIL-052WO1are relatively short-lived with little expansion in vivo, which can help control cytokine release syndrome (CRS) and other adverse events resulting from cytokine expression or CAR armoring cell therapy. In addition, y5 T cells are unlikely to cause graft-versus-host disease (GvHD) as they interact with antigen independent of major histocompatibility complex (MHC)-recognition, permitting use in an allogeneic setting.

[0184] In some embodiments, the cells are human blood-derived y5 T cells.

[0185] In some embodiments, the cells are mouse blood-derived y5 T cells.

[0186] In some embodiments, the cells are rat blood-derived y5 T cells.

[0187] In some embodiments, the cells are non-human mammal blood-derived y5 T cells.

[0188] In some embodiments, the cells are obtained from serum or plasma, wherein the serum or plasma can be obtained from any source including, but not limited to, human peripheral blood, umbilical cord blood, or blood derived from another mammalian species. The plasma may be from a single donor or may be pooled from several donors.

[0189] In some embodiments, the cells are skin-derived y5 T cells.

[0190] In some embodiments, the cells are V51+ y5 T cells.

[0191] The blood-derived y5 T cells primarily express the delta variable 1 (V51) chain.References to “V51 T cells” refer to y5 T cells with a V51 chain, i.e., V51+T cells. As used herein, the term “delta variable 1” may also be referred to as V51 or Vdl.

[0192] In some embodiments, the y5 T cells are V51+(Vdl+) T cells, e.g., human V51+T cells (also referred to as Vdl+T cells).

[0193] In some embodiments, the present invention provides a V51+T cell comprising a CAR described herein. In some embodiments, the V51+T cell may comprise one or more cytokines and / or cytokine receptors. In some embodiments, the V51+T cell may comprise one or more cytokines and / or cytokine receptors in combination with a CAR.

[0194] In some embodiments, the present invention provides a V51+T cell comprising a polynucleotide encoding a CAR described herein. In some embodiments, the V51+T cell may comprise a polynucleotide encoding a one or more cytokines and / or cytokine receptors. In some embodiments, the V51+T cell may comprise a polynucleotide encoding a one or more cytokines and / or cytokine receptors in combination with a CAR.

[0195] In some embodiments, the present invention provides a V51+T cell comprising a CAR described herein. In some embodiments, the V51+T cell may comprise a cytokine or cytokine receptor selected from IL-2 and IL-2Ra / IL-2Rp / IL-2Ry. In some embodiments, the V51+T cell may comprise a cytokine or cytokine receptor selected from IL-2 and IL-2RP, in combination with a CAR.ATTORNEY DOCKET NO. MIL-052WO1

[0196] In some embodiments, the V51+T cell may comprise one or more CARs and IL-2 mutant alone or in combination with IL-2Rp.

[0197] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F according to wild type IL-2.

[0198] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, and Q126T according to wild type IL-2.

[0199] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126H according to wild-type IL-2.

[0200] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126K according to wild type IL-2.

[0201] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126A according to wild type IL-2.

[0202] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126C according to wild type IL-2.

[0203] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, and R38E according to wild type IL-2.

[0204] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and V91T according to wild type IL-2.

[0205] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and Q126E according to wild type IL-2.

[0206] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations of F42A, R38E, and N88T according to wild type IL-2.

[0207] In some embodiments, the V51+T cell comprises one or more CARs and an IL-2 mutein comprising amino acid substations F42A, R38E, and N88R according to wild type IL-2.ATTORNEY DOCKET NO. MIL-052WO1

[0208] In some embodiments, the present invention encompasses a V51+T cell expressing IL-2 comprising any one of SEQ ID NO: 1-11. In some embodiments, the present invention encompasses a V51+T cell expressing IL-2 comprising an amino acid sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-11.

[0209] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% to identical SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 1. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 1.

[0210] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% to identical SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which isATTORNEY DOCKET NO. MIL-052WO198% identical to SEQ ID NO: 2. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 2.

[0211] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 3. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 3.

[0212] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 4. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 4.

[0213] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 5. In some embodiments, the V51+T cellATTORNEY DOCKET NO. MIL-052WO1expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 5. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 5.

[0214] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 6. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 6.

[0215] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 7. In someATTORNEY DOCKET NO. MIL-052WO1embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 7. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 7.

[0216] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% to identical SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 8. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 8.

[0217] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which isATTORNEY DOCKET NO. MIL-052WO196% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 9. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 9.

[0218] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 98% identical to SEQ ID NO: 10. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 10.

[0219] In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 90% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 91% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 92% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 93% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 94% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 95% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 96% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 97% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acidATTORNEY DOCKET NO. MIL-052WO1sequence which is 98% identical to SEQ ID NO: 11. In some embodiments, the V51+T cell expressing IL-2 comprising an amino acid sequence which is 99% identical to SEQ ID NO: 11.

[0220] In some embodiments, the V51+T comprises a wild-type human IL-2. In some embodiments, the V51+T cell has reduced binding to IL-2Rp. In some embodiments, the V51+T cell may also comprise one or more CAR polypeptides.

[0221] In some embodiments, the V51+T cell is engineered to express a CAR and a membrane 11-2 mutein. In some embodiments, the V51+T cell is engineered to express a CAR and a membrane 11-2 mutein and IL-2Rp. In some embodiments, the V51+T cell is engineered to express a CAR and a membrane IL-2 mutein having a signal peptide.

[0222] In some embodiments, the V51+T cell comprise polynucleotides encoding one or more CARs and IL-2 mutant alone or in combination with IL-2Rp. In some embodiments, the V51+T cells comprise a polynucleotide encoding a wild-type human IL-2.Chimeric antigen receptors

[0223] Chimeric Antigen Receptors (CARs) are hybrid molecules comprising three essential units: an extracellular antigen-binding domain, linker / transmembrane motifs, and intracellular signaling motifs. Long el al.. Oncoimmunology, 2013, 2(4): e23621.

[0224] Classic chimeric antigen receptors (CARs) can graft the specificity of, for example, an antibody (Ab) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an extracellular antigen recognizing region, a hinge region, a transmembrane domain all connected to a compound intracellular region which transmits T-cell survival and activation signals. The compound intracellular region generally comprises one or more co-stimulatory domains and a primary signaling / activation domain. The most commonly used primary activation domain to design a CAR polypeptide is a CD3ζ signaling domain.

[0225] CAR modified T cells have been used successfully in the clinic for the treatment of both hematological malignancies (like B cell or granulocyte malignancies) and solid tumors. CAR-T cell-based cancer immunotherapies still need improvement for several important reasons such as cytotoxicity. The present invention develops CARs lacking intracellular primary activation / signaling domain (e.g., intracellular CD3ζ activation domain) with reduced cytotoxicity. The present application specifically provides y5 T cells expressing said CARs in the presence or absence of cytokines. The CARs expressed in y5 T cells (e.g., donor-derived skin y5 T cells) can be used to treat cancer and other immune diseases. y5ATTORNEY DOCKET NO. MIL-052WO1T cells expressing a CAR have functionality comparable to those expressing classic CAR including an intracellular CD3ζ activation signaling domain.

[0226] For example, a chimeric antigen receptor (CAR) fusion protein comprises from N-terminus to C-terminus: (i) an antigen binding domain, (ii) a hinge region; (iii) a transmembrane domain, and (iv) one or more costimulatory domains. In accordance, a CAR polypeptide does not comprise an intracellular signaling / activation domain. Typically, a CAR polypeptide described herein does not comprise an intracellular signaling domain derived from CD3ζ.

[0227] Optionally the CAR expressing cell expresses one or more additional polypeptides. For example, the CAR expresses one or more cytokine receptors. Polynucleotides and vectors encoding these constructs are further provided herein as well as the polypeptides encoded by them in vitro or in vivo.CAR componentsa. CAR Antigen Binding Domain

[0228] CARs can use antigen-binding domain properties to re-direct immune cell specificity and responsiveness to selected targets. The antigen binding domain is the portion of the CAR which recognizes antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain antibody (sdAb); an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor.

[0229] In some embodiments, the antigen binding domain may be derived from an antibody or antigen-binding fragment thereof that binds to the antigen. Examples of antibody fragments include Fab, F(ab')2, single chain variable fragment (scFv), tandem scFv, BiTE, single domain (sdAb) antibody, nanobody, diabody, single chain diabody, minibody, camelid VHH, fusion protein, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), scFv-Fc, multi-specific antibodies formed from antibody fragment; the antibody, or fragment thereof, specific to a disease-associated antigen.

[0230] In some embodiments, the antigen-binding domain may be derived from a polypeptide that binds to a disease associated antigen. In some embodiments, the polypeptideATTORNEY DOCKET NO. MIL-052WO1may be a receptor or a portion of a receptor that binds to an antigen. In another embodiment, the antigen-binding domain may be derived from a ligand that binds to an antigen.

[0231] T he antigen can be a tumor associated antigen (TAA). Exemplary tumor associate antigens may include antigens of 4-IBB, 5 AC, 5T4, A2aR, activin receptor-like kinase 1, AGS-22M6, AKAP4, a-fetoprotein, angiopoietin 2, B7-H3, BAFF, BAGE, BCR-ABL, BORIS, CA-125, CA19-9, C242 antigen, carbonic anhydrase 9 (CA-IX), CCR4, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD28, CD30 (TNFRSF8), CD33, CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 v6, CD51, CD56, CD70, CD71, CD73, CD74, CD79B, CD80, CD137, CD140a, CD152, CD200, CD221, CD274, CEA, ch4D5, CLDN18.2, CS1, CSF1R, CTLA-4, C-X-C chemokine receptor type 4, DLL4, DR5, EBAG9, EGF, EGFR, EGFL7, EpCAM, ERBB2, ERBB3, FAP, fibronectin extra domain-B, folate receptor 1, folate receptor a, folate hydrolase, Frizzled receptor, GAGE, GD2 ganglioside, GD3 ganglioside, glioma, glypican 3, GP MB, gp100, GUCY2C, HER1, HER2 / neu, HER3, HGF, HHGFR, histone complex, HLA-DR, human scatter factor receptor kinase, HPV-16, HSP105, IDH1, IDO1, IGF-I, IGF-1 receptor, ILGF2, IL-6, IL-13, integrin avP3, integrin a5pi, KIR, LAG-3, Lewis-Y antigen, LY6K, MAGE-1, MAGE- A3, MAGE-C2, MAGE-04, MAPG, MART-1, Melan-A, MET, MCP-1, mesothelin, MIF, MSLN (Mesothelin), MS4A1, mucin CanAg, MUC1, MUC4, MUC16, NG2, N-glycolylneuraminic acid, Notch receptor PD-1, NY-ESO-1, OCAA, PAP, PDGF-R a, PDCD1, PD1, PD-L1, phosphatesodium co-transporter, phosphatidylserine, PRAME, PSA, RANKL, RON, R0R1, SDC1, Sialyl-Tn, SLAMF7, SPAG-9, SSX1, STEAP1, survivin, TAG-72, telomerase, TEM1, tenascin C, TGF-P, TFM-3, TLR, TAM, TFM-3, TRAIL-R2, TRAIL-R1, TWEAK receptor, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor antigen CTAA16.88, TYRP1 (glycoprotein 75), VEGF-A, VEGFR2, VEGFR-1, vimentin, VISTA, WT1, and XAGE-lb; the antibody, or fragment thereof, is directed to a disease-associated antigen which is chosen from 1-40-p-amyloid, AOC3 (VAP-1), ACVR2B, angiopoietin 3, P-amyloid, C5, CCL11 (eotaxin-1), CCR5, CD2, CD3, CD4, CD5, CD11, CD 18, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD28, CD41 (integrin a-lib), CD52, CD125, CD147 (basigin), CD 154 (CD40L), CEA-related antigen, clumping factor A, endotoxin, GMCSF receptor a-chain, growth differentiation factor 8, hemagglutinin, HNGF, Hsp90, IGHE, IgEFc region, IL-ip, IL-4, IL-5, IL-6, IL-9, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL-6 receptor, integrin a4p7, integrin a7p7, integrin a4, integrin aI3 / 4p3, interferon a / p receptor, interferon gamma-induced protein, IFN-y, IFN-a, ITGB2 (CD 18), LFA-1 (CD Ila), LINGO- 1, lipoteichoic acid, LOXL2, myelin-associatedATTORNEY DOCKET NO. MIL-052WO1glycoprotein, myostatin, neural apoptosis-regulated proteinase 1, NGF, NOGO-A, Oryctolagus cuniculus, OX-40, PCSK9, phosphatidylserine, platelet-derived growth factor receptor P, RANKL, Rhesus factor, sclerostin, SOST, sphingosine-1 -phosphate, TFPI, TGF-P, TGF P 2, TGF P 1, TNF-a, VEGF-A, and VWF; or the antibody, or fragment thereof.

[0232] In some embodiments, the antigen is a B-cell associated antigen.

[0233] In some embodiments, the antigen is a T-cell associated antigen. For example, the antigen can be selected from CD3, CD4, CD8, PECAM1, CD103 (naive, RTEs); CCR7, CD127, CD62L (Tscm, Tern); IL-2RA (Tern), HLA-DR, CCR5, TBX21, GZMA (Tern, Teff), CCR, ITGAL, IFNg, IL-13, IL-17A, IL-2, IL-21, IL-22, IL-25, IL-26, TBX21, TCF7, EPCAM.

[0234] In some embodiments, the antigen is an antigen associated with a solid tumor.

[0235] In some embodiments, the antigen associated with a tumor is GUCY2C.Guanylyl Cyclase C (GVCY2C or GCC)

[0236] Guanylyl cyclase C (GUCY2C) (also known as STAR, ST Receptor, GUC2C, and GCC) is a transmembrane cell surface receptor that functions in the maintenance of intestinal fluid, electrolyte homeostasis and cell proliferation (Carrithers et al., Proc Natl Acad Sci USA 100: 3018-3020 (2003); Mann et al., Biochem Biophys Res Commun 239: 463-466 (1997); Pitari et al., Proc Natl Acad Sci USA 100: 2695-2699 (2003)); GenBank Accession No. NM — 004963, each of which is incorporated herein by reference). This function is mediated through binding of guanylin (Wiegand et al. FEBS Lett. 311: 150-154 (1992)). GCC also is a receptor for heat-stable enterotoxin which is a peptide produced by E. coli, as well as other infectious organisms (Rao, M. C. Ciba Found. Symp. 112:74-93 (1985); Knoop F. C. and Owens, M. J. Pharmacol. Toxicol. Methods 28:67-72 (1992)). Binding of ST to GCC activates a signal cascade that results in enteric disease, e.g., diarrhea. Nucleotide sequence for human GUC2YC (GenBank Accession No. NM — 004963). The amino acid sequence for human GUCY2C (GenPept Accession No. NP — 004954):MKTLLLDLALWSLLFQPGWLSFSSQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGL E I VRGRLQNAGLNVTVNAT FMYS DGL I HNS GDCRS S TCEGLDLLRKI SNAQRMGCVL I GPS C TYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYS WSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKWLRQDKEFQDILMDHNRKSNVI IM CGGPEFLYKLKGDRAVAEDIVI ILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSF SRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDW GDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQILMI AVFTLTGAWLLLLVALLMLRKYRKDYELRQKKWSHIPPENIFPLETNETNHVSLKIDDDKRATTORNEY DOCKET NO. MIL-052WO1RDTIQRLRQCKYDKKRVILKDLKHNDGNFTEKQKIELNKLLQIDYYNLTKFYGTVKLDTMIF GVIEYCERGSLREVLNDTISYPDGTFMDWEFKISVLYDIAKGMSYLHSSKTEVHGRLKSTNC WDSRMWKITDFGCNSILPPKKDLWTAPEHLRQANISQKGDVYSYGIIAQEIILRKETFYT LSCRDRNEKI FRVENSNGMKPFRPDLFLETAEEKELEVYLLVKNCWEEDPEKRPDFKKIETT LAKIFGLFHDQKNESYMDTLIRRLQLYSRNLEHLVEERTQLYKAERDRADRLNFMLLPRLW KSLKEKGFVEPELYEEVTIYFSDIVGFTTICKYSTPMEWDMLNDIYKSFDHIVDHHDVYKV ETIGDAYMVASGLPKRNGNRHAIDIAKMALEILSFMGTFELEHLPGLPIWIRIGVHSGPCAA GWGIKMPRYCLFGDTVNTASRMESTGLPLRIHVSGSTIAILKRTECQFLYEVRGETYLKGR GNETTYWLTGMKDQKFNLPTPPTVENQQRLQAEFSDMIANSLQKRQAAGIRSQKPRRVASYK KGTLEYLQLNTTDKESTYF (SEQ IDN0: 15)

[0237] The GUCY2C protein has some generally accepted domains each of which contributes to the function of the GUCY2C molecule. GUCY2C functions include a signaling for directing the protein to the cell surface, an extracellular ligand binding, tyrosine kinase activity, and a guanylyl cyclase catalytic activity. In normal human tissues, GUCY2C is expressed at the mucosal cells, e.g., at the apical brush border membranes, lining the small intestine, large intestine and rectum (Carrithers et al., Dis Colon Rectum 39: 171-181 (1996)). GUCY2C expression is maintained upon neoplastic transformation of intestinal epithelial cells, with expression in all primary and metastatic colorectal tumors (Carrithers et al., Dis Colon Rectum 39: 171-181 (1996); Buc et al. Eur J Cancer 41: 1618-1627 (2005); Carrithers et al., Gastroenterology 107: 1653-1661 (1994)). Neoplastic cells from the stomach, esophagus and the gastroesophageal junction also express GUCY2C. See, e.g., U. S. Pat. No.6,767,704; Debruyne et al. Gastroenterology 130:1191-1206 (2006). The tissue-specific expression and association with cancer, e.g., of gastrointestinal origin, (e.g., colon cancer, stomach cancer, or esophageal cancer), can be exploited for the use of GUCY2C as a diagnostic marker for this disease. See Carrithers et al., Dis Colon Rectum 39: 171-181 (1996); Buc et al. Eur J Cancer 41: 1618-1627 (2005).

[0238] As a cell surface protein, GUCY2C can also serve as a therapeutic target for receptor binding proteins such as antibodies or ligands. In normal intestinal tissue, GUCY2C is expressed on the apical side of epithelial cell tight junctions that form an impermeable barrier between the luminal environment and vascular compartment (Almenoff et al., Mol Microbiol 8: 865-873); Guarino et al., Dig Dis Sci 32: 1017-1026 (1987)). As such, systemic intravenous administration of a GUCY2C -binding protein therapeutic will have minimal effect on intestinal GUCY2C receptors, while having access to neoplastic cells of the gastrointestinal system, including invasive or metastatic colon cancer cells, extraintestinal orATTORNEY DOCKET NO. MIL-052WO1metastatic colon tumors, esophageal tumors or stomach tumors, adenocarcinoma at the gastroesophageal junction. Additionally, GUCY2C internalizes through receptor mediated endocytosis upon ligand binding (Buc et al. Eur J Cancer 41: 1618-1627 (2005); Urbanski et al., Biochem Biophys Acta 1245: 29-36 (1995)).

[0239] Polyclonal antibodies raised against the extracellular domain of GUCY2C (Nandi et al. Protein Expr. Purif. 8: 151-159 (1996)) were able to inhibit the ST peptide binding to human and rat GUCY2C and inhibit ST-mediated cGMP production by human GUCY2C.

[0240] GUCY2C has been characterized as a protein involved in cancers, including colon cancers. See also, Carrithers et al., Dis Colon Rectum 39: 171-181 (1996); Buc et al. Eur J Cancer 41: 1618-1627 (2005); Carrithers et al., Gastroenterology 107: 1653-1661 (1994); Urbanski et al., Biochem Biophys Acta 1245: 29-36 (1995).Antigen Binding Molecules of GUCY2C

[0241] In some embodiments, the present invention relates to anti-GUCY2C antigen binding molecules. In some embodiments, anti-GUCY2C molecules of the present inventions cause a cellular reaction upon binding to GUCY2C on a GUCY2C expressing cell to which it binds. In some embodiments, an anti-GUCY2C antigen binding agent can block ligand binding to GUCY2C.

[0242] The naturally occurring mammalian antibody structural unit is typified by a tetramer. Each tetramer is composed of two pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, a, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). The variable regions of each light / heavy chain pair form the antibody binding site. Preferred isotypes for the anti-GUCY2C antibody molecules are IgG immunoglobulins, which can be classified into four subclasses, IgGl, IgG2, IgG3 and IgG4, having different gamma heavy chains. Most therapeutic antibodies are human, chimeric, or humanized antibodies of the IgGl type. In a particular embodiment, the anti-GUCY2C antibody molecule has the IgGl isotype.ATTORNEY DOCKET NO. MIL-052WO1

[0243] The variable regions of each heavy and light chain pair form the antigen binding site. Thus, an intact IgG antibody has two binding sites which are the same. However, bifunctional or bispecific antibodies are artificial hybrid constructs which have two different heavy / light chain pairs, resulting in two different binding sites.

[0244] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989). As used herein, CDRs are referred according to Kabat for each of the heavy (HCDR1, HCDR2, HCDR3) and light (LCDR1, LCDR2, LCDR3) chains.

[0245] An anti-GUCY2C antibody molecule can comprise all, or an antigen binding subset of the CDRs or the heavy chain, of the antibodies described herein. Amino acid sequences of anti-GUCY2C antigen binding agents described herein, including variable regions and CDRs, can be found in Tables 1-3.

[0246] Thus, in an embodiment the antibody molecule includes one or both of:(a) one, two, three, or an antigen binding number of, light chain CDRs (LCDR1, LCDR2 and / or LCDR3) of a human antibody such as an antibody derived from a human hybridoma or a murine antibody (e.g., a light chain of an anti-GUCY2C antibody described in US20180355062A1, which is incorporated by reference in its entirety). In embodiments the CDR(s) may comprise an amino acid sequence of one or more or all of LCDR1-3 as follows: LCDR1, or modified LCDR1 wherein one to seven amino acids are conservatively substituted) LCDR2, or modified LCDR2 wherein one or two amino acids are conservatively substituted); or LCDR3, or modified LCDR3 wherein one or two amino acids are conservatively substituted; and(b) one, two, three, or an antigen binding number of, heavy chain CDRs (HCDR1, HCDR2 and / or HCDR3) as described herein. In embodiments the CDR(s) may comprise an amino acid sequence of one or more or all of HCDR1-3 as follows: HCDR1, or modified HCDR1 wherein one or two amino acids are conservatively substituted; HCDR2, or modified HCDR2 wherein one to four amino acids are conservatively substituted; or HCDR3, or modified HCDR3 wherein one or two amino acids are conservatively substituted.ATTORNEY DOCKET NO. MIL-052WO1

[0247] In some embodiments, an anti-GUCY2C antibody molecule of the invention can draw antibody-dependent cellular cytotoxicity (ADCC) to a cell expressing GUCY2C, e.g, a tumor cell. Antibodies with the IgGl and IgG3 isotypes are useful for eliciting effector function in an antibody-dependent cytotoxic capacity, due to their ability to bind the Fc receptor. Antibodies with the IgG2 and IgG4 isotypes are useful to minimize an ADCC response because of their low ability to bind the Fc receptor. In related embodiments substitutions in the Fc region or changes in the glycosylation composition of an antibody, e.g, by growth in a modified eukaryotic cell line, can be made to enhance the ability of Fc receptors to recognize, bind, and / or mediate cytotoxicity of cells to which anti-GUCY2C antibodies bind. See, e.g., U. S. Pat. Nos. 7,317,091, 5,624,821 and publications including WO 00 / 42072, Shields, et al. J. Biol. Chem. 276:6591-6604 (2001), Lazar et al. Proc. Natl. Acad. Sci. U. S. A. 103:4005-4010 (2006), Satoh et al. Expert Opin Biol. Ther. 6:1161-1173 (2006). In certain embodiments, the antibody or antigen-binding fragment (e.g, antibody of human origin, human antibody) can include amino acid substitutions or replacements that alter or tailor function (e.g, effector function). For example, a constant region of human origin (e.g., yl constant region, y2 constant region) can be designed to reduce complement activation and / or Fc receptor binding. See, e.g., U. S. Pat. No. 5,648,260 (Winter et al.), U. S. Pat. No. 5,624,821 (Winter et al.) and U. S. Pat. No. 5,834,597 (Tso et al.), the entire teachings of which are incorporated herein by reference. Preferably, the amino acid sequence of a constant region of human origin that contains such amino acid substitutions or replacements is at least about 95% identical over the full length to the amino acid sequence of the unaltered constant region of human origin, more preferably at least about 99% identical over the full length to the amino acid sequence of the unaltered constant region of human origin. Additional anti-GUCY2C antigen binding molecules are further described in U. S. Pat. No. 8,785,600 (Nam et al.), the entire teachings of which are incorporated herein by reference.

[0248] In still another embodiment, effector functions can also be altered by modulating the glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody. For example, antibodies with enhanced ADCC activities with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in U. S. Patent Application Publication No. 2003 / 0157108 (Presta). See also U. S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Glycofi has also developed yeast cell lines capable of producing specific glycoforms of antibodies.ATTORNEY DOCKET NO. MIL-052WO1

[0249] Additionally, or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which are engineered to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., (1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

[0250] Humanized antibodies can also be made using a CDR-grafted approach. Techniques of generation of such humanized antibodies are known in the art. Generally, humanized antibodies are produced by obtaining nucleic acid sequences that encode the variable heavy and variable light sequences of an antibody that binds to GUCY2C, identifying the complementary determining region or “CDR” in the variable heavy and variable light sequences and grafting the CDR nucleic acid sequences on to human framework nucleic acid sequences. See, e.g., U. S. Pat. Nos. 4,816,567 and 5,225,539. The location of the CDRs and framework residues can be determined. See Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. J. Mol. Biol. 196:901-917 (1987)).

[0251] Anti-GUCY2C antibody molecules described herein have the CDR amino acid sequences and nucleic acid sequences encoding CDRs listed in Table 4. In some embodiments, sequences from Table 4 can be incorporated into molecules which recognize GUCY2C for use in the therapeutic or diagnostic methods described herein. The human framework that is selected is one that is suitable for in vivo administration, meaning that itATTORNEY DOCKET NO. MIL-052WO1does not exhibit immunogenicity. For example, such a determination can be made by prior experience with in vivo usage of such antibodies and studies of amino acid similarities. A suitable framework region can be selected from an antibody of human origin having at least about 65% amino acid sequence identity, and preferably at least about 70%, 80%, 90% or 95% amino acid sequence identity over the length of the framework region within the amino acid sequence of the equivalent portion (e.g., framework region) of the donor antibody, e.g., an anti-GUCY2C antibody molecule (e.g., 3G1). Amino acid sequence identity can be determined using a suitable amino acid sequence alignment algorithm, such as CLUSTAL W, using the default parameters. (Thompson J. D. et al., Nucleic Acids Res. 22:4673-4680 (1994).)

[0252] Once the CDRs and FRs of the cloned antibody that are to be humanized are identified, the amino acid sequences encoding the CDRs are identified and the corresponding nucleic acid sequences grafted on to selected human FRs. This can be done using known primers and linkers, the selection of which are known in the art. All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen. After the CDRs are grafted onto selected human FRs, the resulting “humanized” variable heavy and variable light sequences are expressed to produce a humanized Fv or humanized antibody that binds to GUCY2C. Preferably, the CDR-grafted (e.g., humanized) antibody binds a GUCY2C protein with an affinity similar to, substantially the same as, or better than that of the donor antibody. Typically, the humanized variable heavy and light sequences are expressed as a fusion protein with human constant domain sequences so an intact antibody that binds to GUCY2C is obtained. However, a humanized Fv antibody can be produced that does not contain the constant sequences.

[0253] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, humanized antibodies can have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR. See, e.g., U. S. Pat. Nos. 5,585,089 or 5,859,205. The acceptor framework can be a mature human antibody framework sequence or a consensus sequence. As used herein, the term “consensus sequence” refers to the sequence found mostATTORNEY DOCKET NO. MIL-052WO1frequently, or devised from the most common residues at each position in a sequence in a region among related family members. A number of human antibody consensus sequences are available, including consensus sequences for the different subgroups of human variable regions. See Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, U. S. Government Printing Office (1991). The Kabat database and its applications are freely available online, e.g., via IgBLAST at the National Center for Biotechnology Information, Bethesda, Md. See also Johnson, G. and Wu, T. T., Nucleic Acids Research 29:205-206 (2001).

[0254] In certain embodiments, the GUCY2C antibody molecule is a human anti-GUCY2C IgGl antibody. Since such antibodies possess desired binding to the GUCY2C molecule, any one of such antibodies can be readily isotype-switched to generate a human IgG4 isotype, for example, while still possessing the same variable region (which defines the antibody's specificity and affinity, to a certain extent). Accordingly, as antibody candidates are generated that meet desired “structural” attributes as discussed above, they can generally be provided with at least certain additional “functional” attributes that are desired through isotype switching.

[0255] In some embodiments, the portion of a CAR composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.

[0256] In this way, FR residues can be selected and combined from the recipient and import sequences that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0257] A humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present invention, the ability to bind human GUCY2C. InATTORNEY DOCKET NO. MIL-052WO1some embodiments, a humanized antibody or antibody fragment may have improved affinity and / or specificity of binding to human GUCY2C.

[0258] In some embodiments, the anti-GUCY2C antigen binding agent comprises one or more CDR sequences provided in Table 1. In some embodiments, the anti-GUCY2C antigen binding agent comprises a heavy chain variable region with a CDR 1 provided in Table 1. In some embodiments, the anti-GUCY2C antigen binding agent comprises a heavy chain variable region with a CDR 2 provided in Table 1. In some embodiments, the anti-GUCY2C antigen binding agent comprises a heavy chain variable region with a CDR 3 provided in Table 1. In some embodiments, the anti-GUCY2C antigen binding agent comprises a heavy chain variable region with a CDR1, CDR2, and CDR3 provided in Table 1. In some embodiments, the anti-GUCY2C antigen binding agent comprises one or more CDR sequences provided in Table 1 wherein said CDR comprises 1, 2, or 3 amino acid substitutions. In one embodiment, said substitution does not adversely affect the binding of the binding agent to its target.Table 1. Exemplary Anti-GUCY2C CDR sequences according to KabatSingle Domain Antibodies of GUCY2C

[0259] Single-domain antibodies (sdAbs) are different from conventional 4-chain antibodies by having a single monomeric antibody variable domain. For example, cam elids and sharks produce sdAbs named heavy chain-only antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of the camelid heavy-chain only antibodies has a single heavy chain variable domain (VHH), which can have high affinity to an antigen without the aid of a light chain. Camelid VHH is known as the smallest functional antigenbinding fragment with a molecular weight of approximately 15 kDa. In some embodiments, the antigen binding agents are single human heavy chain variable domain (VH) antibodies. Such binding molecules are also termed Humabody® and may be used interchangeably herein. Humabody® is a registered trademark of Crescendo Biologies Ltd.ATTORNEY DOCKET NO. MIL-052WO1

[0260] One aspect of the present application provides isolated single-domain antibodies (referred herein as “anti-GUCY2C sdAbs”) that specifically bind to GUCY2C, such as human GUCY2C. In some embodiments, the anti-GUCY2C sdAb modulates GUCY2C activity. In some embodiments, the anti-GUCY2C sdAb is an antagonist antibody. Further provided are antigen-binding fragments derived from any one of the anti-GUCY2C sdAbs described herein, and antigen binding proteins comprising any one of the anti-GUCY2C sdAbs described herein. In some embodiments, the anti-GUCY2C sdAb comprise one, two and / or three CDR sequences provided in Table 1. Exemplary anti-GUCY2C sdAbs are listed in Table 2 and Table 3. In some embodiments, the anti-GUCY2C sdAb comprises a variable heavy chain provided in Table 2 or Table 3.

[0261] In some embodiments, some or all of the CDRs sequences, the heavy chain, can be used in another antigen binding agent, e.g, in a CDR-grafted, humanized, or chimeric antibody molecule. Embodiments include an antibody molecule that comprises sufficient CDRs, e.g, all three CDRs from one of the above-referenced heavy chain variable region, to allow binding to cell surface GUCY2C.

[0262] In some embodiments the CDRs, e.g., all of the HCDRs, are embedded in human or human derived framework region(s). Examples of human framework regions include human germline framework sequences, human germline sequences that have been affinity matured (either in vivo or in vitro), or synthetic human sequences, e.g., consensus sequences. In an embodiment the heavy chain framework is an IgGl or IgG2 framework.

[0263] In some embodiments, the anti-GUCY2C antigen binding agents of the present invention comprise a heavy chain variable region amino acid sequence provided in Table 2. In some embodiments, the anti-GUCY2C antigen binding agents are single domain heavy chain only antibodies (e.g., antigen binding agents that do not comprise an immunoglobulin light chain).

[0264] Table 2. Exemplary Heavy Chain Variable Region (VH) Amino Acid SequencesATTORNEY DOCKET NO. MIL-052WO1Table 3. Exemplary Heavy Chain Variable Region (VH) Amino Acid Sequencesb. CAR Transmembrane Domain

[0265] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the antigen-binding domain of the CAR, e.g., through a hinge sequence. The transmembrane connects the intracellular signaling domain to the hinge region of a CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.ATTORNEY DOCKET NO. MIL-052WO1

[0266] A suitable transmembrane domain of particular use in an CAR described herein may be a transmembrane domain derived from CD28, 4-1BB / CD137, CD8 (e.g., CD8a), CD4, CD19, CD3 epsilon, CD3 zeta, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137,, CD 154, CTLA4, PD-1, CD 154, TCR a, or TCR 0, and / or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. Exemplary transmembrane domains are disclosed in WO 2020 / 227446; which is incorporated herein in its entirety.

[0267] Alternatively, the transmembrane domain in a CAR described herein may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A transmembrane domain of the invention is thermodynamically stable in a membrane. It may be a single a helix, a transmembrane 0 barrel, a 0-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length.

[0268] In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used in addition to the transmembrane domains described supra. In some embodiments, the transmembrane domain can be selected by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0269] Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In some embodiments, the linker is a glycine-serine doublet or a triple alanine linker.

[0270] In some embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used in addition to the transmembrane domains described supra. In some embodiments, the transmembrane domain can be selected by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.ATTORNEY DOCKET NO. MIL-052WO1

[0271] In some embodiments, the transmembrane domain in the CAR of the invention is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises the nucleic acid sequence of FWVLVWGGVLACYSLLVTVAFI I FWV (SEQ ID NO: 45). In some embodiments, the CD28 transmembrane domain comprises a nucleic acid sequence that encodes the amino acid sequence of the CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of the CD28 transmembrane domain or a sequence with at least 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of FWVLVWGGVLACYSLLVTVAFI I FWV (SEQ ID NO: 45). In some embodiments, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.c. CAR Hinge Domain

[0272] A CAR molecule generally includes the structure of the spacer / hinge domain between the antigen binding domain and the transmembrane domain. The hinge domain is a spacer that provides separation of the antigen binding domain (e.g., scFv) from the cell membrane and an intracellular signaling module that mediates T-cell activation. One of ordinary skill in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates flexibility. See, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004). The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule such as a human protein. In some embodiments, the length of the hinge sequence may be optimized based on the CAR and targeted antigens.

[0273] A CAR contemplated herein comprises a hinge sequence between the antigenbinding domain and the transmembrane domain.

[0274] In some embodiments, the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the spacer domain includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the hinge domain may also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge isATTORNEY DOCKET NO. MIL-052WO1derived from or includes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgGl Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino acid substitutions, modifications, or deletions that cause impaired binding of the hinge domain to an Fc receptor (FcR).

[0275] In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises the nucleic acid sequence ofIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20). In some embodiments, the hinge domain comprises a nucleic acid sequence that encodes the amino acid sequence of IEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20). In some embodiments, the hinge domain comprises a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of, or a sequence with 95-99% identity to, an amino acid sequence of IEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 20).

[0276] In some embodiments, the CAR comprises a CD8 hinge domain. In some embodiments the hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 71). In some embodiments the CAR comprises a CD8 transmembrane domain. In some embodiments the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 71). In some embodiments, the hinge and transmembrane domains are derived from the same molecule. In other embodiments, the hinge and transmembrane domains are derived from different molecules (e.g., CD8 fused to CD28). In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises the amino acid sequence ofIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI I FWV (SEQ ID NO: 72). In some embodiments, the hinge domain comprises a nucleic acid sequence that encodes the amino acid sequence of the hinge domain. In some embodiments, the hinge domain comprises a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence ofATTORNEY DOCKET NO. MIL-052WO1IEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWV (SEQ IDNO: 72).d. CAR Intracellular Signal Domain

[0277] The intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0278] Examples of intracellular signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0279] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. In some embodiments, the ITAM-containing domain within the CAR recapitulates the signaling of the primary TCR independently of endogenous TCR complexes. In one aspect, the primary signal is initiated by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to,ATTORNEY DOCKET NO. MIL-052WO1proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or IT AM.

[0280] Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 Zeta), FcR gamma, FcR P, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples, of the IT AM include peptides having sequences of amino acid numbers 51 to 164 of CD3.zeta. (NCBIRefSeq: NP.sub.--932170.1), amino acid numbers 45 to 86 of Fc.epsilon. RI.gamma. (NCBIRefSeq: NP. sub.— 004097.1), amino acid numbers 201 to 244 of Fc. epsilon. RI.p. (NCBI RefSeq: NP. sub. —000130.1), amino acid numbers 139 to 182 of CD3. gamma. (NCBI RefSeq: NP. sub.— 000064.1), amino acid numbers 128 to 171 of CD3. delta. (NCBI RefSeq: NP. sub. —000723.1), amino acid numbers 153 to 207 of CD3. epsilon. (NCBI RefSeq: NP. sub. —000724.1), amino acid numbers 402 to 495 of CD5 (NCBIRefSeq: NP.sub. -055022.2), amino acid numbers 707 to 847 of 0022 (NCBI RefSeq: NP.sub. -001762.2), amino acid numbers 166 to 226 of CD79a (NCBI RefSeq: NP.sub.— 001774.1), amino acid numbers 182 to 229 of CD79b (NCBIRefSeq: NP.sub.— 000617.1), and amino acid numbers 177 to 252 of CD66d (NCBI RefSeq: NP.sub.— 001806.2), and their variants having the same function as these peptides have. The amino acid number based on amino acid sequence information of NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor (comprising a signal peptide sequence etc.) of each protein.

[0281] In embodiments, the intracellular signal domain transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0282] The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell. Examples of immune effector function, e.g., in a CART cell, include cytolytic activity and helper activity, including the secretion of cytokines.ATTORNEY DOCKET NO. MIL-052WO1

[0283] In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CAR-T, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from coreceptor or costimulatory molecule.

[0284] In some embodiments, the primary cytoplasmic signaling sequences include, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), FceRI, CD66d, DAP10 and DAP12. In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0285] In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0286] In some embodiments, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. In some embodiments, the costimulatory domainATTORNEY DOCKET NO. MIL-052WO1comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla / CD18) and 4-1BB (CD137)

[0287] Specific, non-limiting examples, of such costimulatory molecules include peptides having sequences of amino acid numbers 236 to 351 of CD2 (NCBI RefSeq: NP.sub.— 001758.2), amino acid numbers 421 to 458 of CD4 (NCBI RefSeq: NP.sub. -000607.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: NP.sub.— 055022.2), amino acid numbers 207 to 235 of CD8.a. (NCBI RefSeq: NP.sub.— 001759.3), amino acid numbers 196 to 210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI RefSeq: NP.sub. -006130.1), amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI RefSeq: NP.sub. -001552.2), amino acid numbers 241 to 277 of CD134 (0X40, NCBI RefSeq: NP.sub.— 003318.1), and amino acid numbers 166 to 199 of ICOS (NCBI RefSeq: NP.sub.— 036224.1), and their variants having the same function as these peptides have. Thus, while the disclosure herein is exemplified primarily with 4- IBB as the co-stimulatory signaling element, other costimulatory elements are within the scope of the disclosure.

[0288] The intracellular signaling domain of the CAR can comprise the primary signaling domain, e.g., CD3-zeta signaling domain, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the CAR intracellular signaling domain can comprise a primary signaling domain, e.g., CD3 zeta chain portion, and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3): 696-706). The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention may be linked to each other in a random or specified order.e. CAR Costimulatory Domain

[0289] The cytoplasmic domains of a classic CAR construct trigger or elicit activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. A classic CAR generally comprises one or more costimulatory domain and a primary activation / signaling domain which is a CD3ζ signaling domain in most CARs.

[0290] A CAR of the present invention does not comprise a primary intracellular activation / signaling domain. In one embodiment, the CAR of the present invention does not comprise a CD3ζ signaling domain.ATTORNEY DOCKET NO. MIL-052WO1

[0291] In accordance, a CAR may comprise one or more costimulatory domains. As used herein, the term “costimulatory signaling domain,” or “costimulatory domain”, refers to an intracellular signaling domain derived from a co-stimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T cells upon binding to antigen. Costimulatory signals are required to achieve robust chimeric antigen receptor (CAR) comprising cell expansion, function, persistence and antitumor activity.

[0292] In some embodiments, a costimulatory region according to the present invention is a signaling region of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), immune cell function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig a (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8a, CD80, IL-2R 0, IL-2R gamma, IL7R a, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0293] In some cases, the design of a CAR may be optimized in light of the y5 TCR molecular structure and costimulation. The costimulatory domains promotes survival, proliferation and activation of y5 T cells.

[0294] In some embodiments, the CAR intracellular domain is designed to comprise a CD28 costimulatory signaling domain. In some embodiments, the CAR intracellular domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 48).ATTORNEY DOCKET NO. MIL-052WO1

[0295] In some embodiments, the CAR intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In some embodiments, the CAR intracellular domain comprises a CD3-zeta with one or more modified immunoreceptor tyrosine based-activation motifs (IT AMs). In some embodiments, the CAR intracellular domain comprises a CD3-zeta with the first of the three immunoreceptor tyrosine based-activation motifs (IT AMs) unmodified and the second and third IT AMs altered, named “1XX”. In some embodiments, the CAR intracellular domain of the CAR comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to that amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNEL QKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 49).

[0296] In another embodiment, the CAR intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB. In yet another embodiment, the CAR intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

[0297] In some embodiments, the CAR intracellular domain is designed to comprise the signaling domain of 4- IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4- IBB comprises and amino acid sequenceKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 18) and the signaling domain of CD3-zeta comprises the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19) or the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 73).

[0298] In some embodiments, the CAR intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.ATTORNEY DOCKET NO. MIL-052WO1

[0299] In some embodiments, the CAR intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB.

[0300] In some embodiments, the CAR intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In one aspect, the intracellular is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In In one aspect, the intracellular is designed to comprise the signaling domain of CD3-zeta and the signaling domain of ICOS.

[0301] In some embodiments, the anti-GCC CAR comprises a GCC binder comprising 3 CDRs of GCC, GCC1, GCC2, GCC3, or GCC4 in Table 1. In some embodiments, the anti-GCC CAR comprises a GCC binder comprising a VHH sequence in Tables 2 and 3.

[0302] In some embodiments, the anti-GCC CAR comprises a GCC binder, a CD28 hinge, a CD28 transmembrane, a 4-1BB co-stimulation and a CD3z activation domain. In some embodiments, the anti-GCC CAR comprises a GCC binder, a CD28hinge, a CD28 transmembrane, a CD28 co-stimulation and a CD3z activation domain.

[0303] In some embodiments, the anti-GCC CAR comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence provided in Table 4.

[0304] Examples of anti-GUC2Y CARs are described in WO 2022 / 123316, which is incorporated herein by reference for all purposes.f. Additional Polypeptides

[0305] In some embodiments, a CAR polypeptide further expresses one or more additional polypeptides. As non-limiting examples, the CAR polypeptide comprises a cytokine receptor. Cytokine regulation is critical for efficient skin yS T cell localization, homeostatic turnover, and downstream function in the skin. For example, IL -2 and IL-7 are required for skin y§ T cell proliferation.g. Linker

[0306] In some embodiments, the additional polypeptides can be linked to the CAR fusion protein (for example, to the costimulatory domain) using a linker.

[0307] In some embodiments, the linker is a peptide linker. A peptide linker refers to a plurality of amino acid residues between the various polypeptide domains added for appropriate spacing and conformation of the molecule. In some embodiments, a linkerATTORNEY DOCKET NO. MIL-052WO1sequence separates one or more heavy or light chain variable domains of an antigen binding domain, hinge domains, transmembrane domains, co-stimulatory domains, and / or additional polypeptides. Exemplary linkers suitable for use in particular embodiments contemplated herein include GGGS, GGS or GS repeats.

[0308] The linking motifs of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or designed to be an extended flexible linker. In some embodiments, the anti-GUCY2C CAR includes a (Gly4-Ser)n (SEQ ID NO: 13) linker, wherein n is 1, 2, 3, 4, 5, or 6. In some embodiments, the linker comprises the amino acid sequence RAAA (SEQ ID NO: 14).

[0309] In some embodiments, the linker is a cleavable linker. In some embodiment, the linker is a non-cleavable linker.

[0310] In some embodiments, the linker is a self-cleaving peptide, e.g., 2A peptides. 2A peptides are a class of 18-22 aa-long peptides. Exemplary 2A peptides include P2A, T2A, E2A and F2A.Switch Receptors

[0311] In some embodiments, the present invention also encompasses immune cells comprising chimeric switch receptors (also referred herein as “switch receptors”). In some embodiments, immune cells that are genetically engineered to express a chimeric switch receptor (CSR) composed of the extracellular ligand binding domain of the human inhibitory receptor fused to the transmembrane and cytoplasmic co-stimulatory signaling domains of with potential immunomodulating and antineoplastic activities.

[0312] Major adverse events associated with CAR- / TCR-T cell therapy include cytokine release syndrome (CRS), neurotoxicity, and on-target off-tumor toxicity, while graft-versus-host-disease (GVHD) forms a major limitation of donor immune cell infusion (DLI). The scarcity of truly tumor-selective antigens, and hence the risk of on-target off-tumor toxicity, forms a more fundamental issue, in particular for solid tumors. With the aim to allow control over infused cell products while sparing host immune activity, a number of safety switch systems has been developed. Early examples of safety switches that irreversibly inactivate infused T cell populations have been used in DLI therapies, where occurrence of graft-versus-host-disease (GVHD) is correlated with infused T cell numbers. Specifically, the efficacy of an HSV-TK-based suicide switch for the treatment of GVHD upon donor immune cell infusion has been well established in clinical studies.ATTORNEY DOCKET NO. MIL-052WO1

[0313] In some embodiments, switch receptors comprise receptors which can turn on / off target toxicities as well as a cytokine release syndrome (reviewed by Tey Clin. Transl.Immunol., 2014, 3: e 17 10.1038).

[0314] Development of a tunable switch that can turn on or off the transgenic immunotherapeutic agent expression is needed in case of adverse events. For example, adoptive cell therapies may have a very long and an indefinite half-life. Since toxicity can be progressive, a safety switch is desired to eliminate the infused cells. Systems and methods that can tune the transgenic protein level and expression window with high flexibility can enhance therapeutic benefit and reduces potential side effects.

[0315] In some embodiments, the switch can be selected from a Caspase 9, an inducible FAS (iFAS), an inducible Caspase 9 (iCasp9), a CD20 / anti-CD20 antibody pair, a protein tag / anti-tag antibody, and a compact suicide gene (RQR8).Table 4. Exemplary SequencesATTORNEY DOCKET NO. MIL-052WO1I IL2AE88T APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEE § IL-2 Mutein ELKPLEEVLNLAQSKNFHLRPRDLISNITVIVLELKGSETTFMCEYADETATIVEFLNRWITF |CQSIISTLT | 1 IL2AE88R APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEE § §ATTORNEY DOCKET NO. MIL-052WO1ATTORNEY DOCKET NO. MIL-052WO1ATTORNEY DOCKET NO. MIL-052WO1ATTORNEY DOCKET NO. MIL-052WO1| §§ § § § § §§ § § § § ||ATTORNEY DOCKET NO. MIL-052WO1GAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCAC | CCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGA | GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT | CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC | GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG | GCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGG | ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCA | GTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTA | A § CC3 CAR ATGGAGCTGGGATTGTCCTGGGTTTTCCTGGTGGCTATACTCGAAGGCGTACAGTGT § ucleic acid GAAGTGCAGTTGGTGGAGAGTGGCGGTGGCCTGGCCCAGCCGGGAGGCTCTTTGA § equence GACTCTCCTGCGCTGCCTCCGGCTTCACTTTCTCCCGCTATTGGATGACCTGGGTCCG §GCAGGCGCCCGGCGGACGCCTGGAGTGGGTGGCTAAGATCAAGTATGATGGATCA § GAAAAATATTACGCAGATAGCGTAAAAGGCCGGTTCACAATATCCAGGGATAATGC § AAAAAACTCCCTGTATCTGCAGATGGATAGCCTGCGCGCTGAAGACACCGCCGTATA § TTATTGCACAAGAGACTACAATAAAGATTACTGGGGCCAGGGAACCCTGGTTACGGT § GAGCTCACGGGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGA § GAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCT | ATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC | TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAG | GAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCAC | CCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGA | GTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT | CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGAC | GTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG | GCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGG | ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCA | GTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC | CC4 CAR ATGGAGCTGGGGCTTTCTTGGGTGTTTCTGGTAGCCATCCTCGAGGGAGTCCAGTGC § ucleic acid GAGGTCCAGCTCGTCGAATCTGGCGGGGGGCTGGTCCAGCCTGGCGGTTCTCTCCG § equence CCTGACCTGTGCGGCCTCAGGGTTCACTTTCAGCCGGTACTGGATGACATGGGTGAG §ACAGGCCCCCGGCAAGGGACTGGAATGGGTAGCAAAGATTAGGCACGACGGCGGT § GAGAAATACTATCCCGACAGTGTCAAGGGGCGGTTTACTGTCTCCCGAGATAATGCC § AAAAACTCACTCTACCTGCAGATGGATAATCTGCGAGCGGAGGATACTGCTATGTAC § TACTGTACTCGAGACTACAACAAGGACCTGTGGGGGCAGGGGACACTGGTGACGGT § TAGTTCTCGGGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAG § AAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTA § TTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT § GCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGA § GCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC § GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGT § GAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCT § ATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGT § GGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC § CTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGAT § GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCAGT | ACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC § GCC CAR ATGGAATTGGGCCTGAGCTGGGTCTTCTTGGTGGCAATCCTGGAGGGCGTGCAGTG ]CGAGGTTCAATTGGTGGAGTCCGGAGGTGGACTGGTGCAACCCGGAGGTAGTCTGA § Nucleic acid GGCTGTCATGTGCCGCCTCTGGGTTCACCTTTTCTCGGTATTGGATGACCTGGGTTAG § sequence ACAGGCCCCCGGCAAAGGTCTTGAGTGGGTAGCAAAAATCAGACACGATGGCGGG §ATTORNEY DOCKET NO. MIL-052WO1

[0316] Anti-GUCY2C antibodies that are not intact antibodies are also useful in this invention. Such antibodies may be derived from any of the antibodies described above.Useful antibody molecules of this type include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544- 546 (1989)), which consists of a VH domain; (vii) a single domain functional heavy chain antibody, which consists of a VHH domain (known as a nanobody) (see, e.g., Cortez-Retamozo, et al., Cancer Res. 64: 2853-2857 (2004), and references cited therein); and (vii) an isolated CDR, e.g., one or more isolated CDRs together with sufficient framework to provide an antigen binding fragment. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). See, e.g., Bird et al. Science 242:423-426 (1988); and Huston et al. Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.ATTORNEY DOCKET NO. MIL-052WO1Antibody fragments, such as Fv, F(ab')2 and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage.Polynucleotides and Vectors

[0317] In some embodiments, the present invention provides a polynucleotide comprising a nucleotide sequence encoding an IL-2 mutein described herein. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an IL-2 mutein of any one of SEQ ID NOs: 1-11. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide and an IL-2 mutein of any one of SEQ ID NOs: 1-11. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide, an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain, and a cellular signal domain.. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain derived from CD8.. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain derived from CD8 and a cellular signal domain. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide, an IL-2 mutein of any one of SEQ ID NOs: 1-11, a hinge domain and transmembrane domain derived from CD8.

[0318] In some embodiments, the present invention provides a polynucleotide encoding a CAR described herein.

[0319] In some embodiments, the polynucleotide is in a viral vector such as a AAV vector, or a non-viral vector such as a plasmid.Cell Culturing and expanding methods

[0320] In another aspect, the present invention provides a composition comprising immune cells as discussed herein. The immune cells are engineered to express an IL-2 mutein described herein. In some embodiments, the immune cells are further engineered to express one or more chimeric antigen receptors (CARs). In some embodiments, the immune cells are further engineered to express a TCR. Any method known in the art can be used to transfect or infect immune cells for expressing an IL-2 mutein and / or a CAR, or a TCR. For example, anATTORNEY DOCKET NO. MIL-052WO1immune cell, e.g., a T cell or a NK cell is transfected with a vector comprising a polynucleotide encoding an IL-2 mutein described herein, and / or a polynucleotide encoding a CAR.

[0321] In some embodiments, the present immune cells are expanded to prepare a cell composition, e.g., a cell composition for therapeutic application (cell therapy). As used herein, the term "expansion" or "proliferation" refers to cell growth and multiplication of cell numbers. Expansion or proliferation, as used herein relate to increased numbers of immune cells (e.g., yd T cells and NK cells) occurring during the culturing process as disclosed herein.Culturing and expanding V61 yd T cells

[0322] In some embodiments, V51+y5 T cells can be obtained using any methods known in the art. In some embodiments, the V51+y5 T cells described here are prepared using a V51+y5 T cell-biased expansion protocol with the differentiation of cytotoxic effector cells expressing high levels of NKRs (e.g., NKp46 and NKG2D) (Almeida et al., Clin Cancer, 2016, 22, 5795-5804; and Correia et al., Blood, 2011, 118, 992-1001; the contents of each of which are incorporated herein by reference in their entirety).

[0323] In some embodiments, V51+ y5 T cells are from ex vitro expansion of V51+ T cells from healthy donors, for example, isolated peripheral blood V51+ y5 T cells. In some embodiments, the V51+ y5 T cells from healthy donors are expanded and screened to establish a master cell bank (MCB), which is the basis for allogeneic cell therapy. For example, the V51+ y5 T cells described are “off-the-shelf’ supplies for treating multiple patients.

[0324] In some embodiments, V51+ y5 T cells are isolated and expanded and differentiated from peripheral blood mononuclear cells (PBMCs). For example, these cells that are expanded and differentiated from PBMCs can be described herein as blood-derived V51+ y5 T cells.

[0325] In other embodiments, V51+ y5 T cells are isolated and expanded from non-hematopoietic tissues. The isolated non-hematopoeitic tissues are cultured in the presence of one or more cytokines selected from interleukin-2 (IL-2), interleukin- 15 (IL- 15), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-21 (IL-21) and combinations thereof.

[0326] In some embodiments, V51+ y5 T cells are produced using a 2-step culture method described in US Patent No. US 11166983; the contents of which are incorporated herein by reference in their entirety.ATTORNEY DOCKET NO. MIL-052WO1

[0327] In some embodiments, V51+ y5 T cells are obtained from a cell preparation sample by a method comprising culturing the sample in a medium comprising a T cell mitogen and a growth factor having interleukin-4-like activity, in the absence of a growth factor having interleukin- 15-like activity. In some examples, the growth factor having interleukin-4-like activity is interleukin-4 (IL-4).

[0328] In other embodiments, V51+ y5 T cells are obtained from a cell preparation sample by a method comprising culturing the sample in a medium comprising a T cell mitogen and a growth factor having interleukin-15-like activity, in the absence of a growth factor having interleukin-4-like activity. In some examples, the growth factor having interleukin- 15-like activity is either interleukin- 15 (IL- 15), interleukin-2 (IL-2), or interleukin-7 (IL-7). As a non-limiting example, the growth factor having interleukin- 15-like activity is IL-15.

[0329] In yet other embodiments, V51+ y5 T cells are obtained from a cell preparation sample by a method comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and a growth factor having interleukin-4-like activity; in the absence of a growth factor having interleukin-15-like activity; and (2) culturing the cells obtained in step (1) in a second culture medium comprising a T cell mitogen and a growth factor having interleukin- 15-like activity, in the absence of a growth factor having interleukin-4-like activity. In some examples, the growth factor having interleukin-4-like activity is interleukin-4 (IL-4) and the growth factor having interleukin- 15-like activity is either interleukin- 15 (IL- 15), interleukin-2 (IL-2), or interleukin-7 (IL-7). As anon-limiting example, the growth factors are IL-4 and IL-15.

[0330] In yet other embodiments, V51+ y5 T cells are obtained from a cell preparation sample by a method comprising: (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and a growth factor having interleukin-4-like activity; in the absence of a growth factor having interleukin-15-like activity; and (2) culturing the cells obtained in step (1) in a second culture medium comprising a growth factor having interleukin- 15-like activity, in the absence of a growth factor having interleukin-4-like activity. In some examples, the growth factor having interleukin-4-like activity is interleukin-4 (IL-4) and the growth factor having interleukin- 15-like activity is either interleukin- 15 (IL-15), interleukin-2 (IL-2), or interleukin-7 (IL-7). As anon-limiting example, the growth factors are IL-4 and IL-15.Culturing and expanding NK cells

[0331] In some embodiments, NK cells are purified from a human blood sample such as cord blood, using any known methods, for example, by positive magnetic cell separation. In someATTORNEY DOCKET NO. MIL-052WO1embodiments, NK cells are purified from a human blood sample by removal of T cells by use of magnetic beads coupled to an anti-CD3 antibody or fragment thereof and further purification by CD56 enrichment. NK cell isolation and NK cell cultivation is performed in a closed system as known by the person skilled in the art.

[0332] The separated NK cell can be further enriched using antibodies that recognize NK cell specific markers. An enriched population of NK cells is added to a cell culture medium suitable for expansion of NK cells, i.e. the medium comprises IL-2 and / or IL-15 and B-cell derived feeder cells such as EBV-LCL. The NK cells can be cultured as long as required to reach the desired amount of expanded NK cells and the produced NK cells can be harvested.

[0333] In some embodiments, the method forthe expansion of NK cells comprises coculturing, in a culture media, a population of NK cells with a feeder cell population, supplementing the culture media with interleukin 2 (IL2) and supplementingthe culture media with at least one soluble stimulatory agent selected from interleukin 12 (IL 12), interleukin 18 (IL 18), interleukin 21 (IL21 ), and combinations thereof. In some embodiments, the feeder cell population comprises cells engineered to express 4- 1 BBL and membrane-bound interleukin- 15 (mblL15).

[0334] In some embodiments, the NK cells are purified and expanded but not limited for NKG2C positive adaptive NK cell subpopulations, CD57 positive subpopulation, and a cytokine secreting subpopulation.

[0335] In some embodiments, the NK cells expanded are a population of NK cells which are genetically modified. In some embodiments, the genetically modified NK cells express a chimeric antigen receptor (CAR) or a TCR of the present invention. In some embodiments, the genetically modified NK cells express an IL-2 mutein of the present invention. In some embodiments, the genetically modified NK cells express a chimeric antigen receptor (CAR) and an IL_2 mutein of the present invention.

[0336] In some embodiments, the genetically modified NK cells express a TCR and an IL_2 mutein of the present invention.Compositions and formulations

[0337] In yet another aspects, the present invention provides a cell composition comprising a population of immune cells described herein. In some embodiments, the composition is a pharmaceutical composition.ATTORNEY DOCKET NO. MIL-052WO1

[0338] In some embodiments, the cell composition comprises a population of NK cells expressing an IL-2 mutein of the present invention. In some embodiments, the cell composition comprises a population of NK cells expressing a CAR and an IL-2 mutein of the present invention. In some embodiments, the cell composition comprises a population of NK cells expressing a TCR and an IL-2 mutein of the present invention.

[0339] In some embodiments, the cell composition comprises a population of y5 T cells expressing an IL-2 mutein of the present invention. In some embodiments, the cell composition comprises a population of y5 T cells expressing a CAR and an IL-2 mutein of the present invention. As non-limiting examples, the cell composition comprises a population of y5 T cells expressing a anti-GCC CAR and an IL-2 mutein.

[0340] In some embodiments, the cell composition is formulated for cryopreservation.

[0341] In some embodiments, the cell composition is cell therapy.

[0342] In some aspects, the cell compositions are the therapeutic compositions (or therapeutic combinations) formulated for intravenous (IV), intrathecal, intramuscular (IM), intraperitoneal (IP) or intratumoral (IT) administration. In some embodiments, the cell composition is formulated in a unit dosage form.

[0343] Also provided herein in certain aspects are pharmaceutical compositions containing any of the compositions provided herein and a pharmaceutically acceptable carrier.Methods of Treatment

[0344] In accordance with present invention, cytokines, armored CARs, polynucleotides or vectors for the same, compositions and modified y5 T cells or CB-NK cells comprising one or more cytokines, CARs described herein can be used for immunotherapy, for example, increasing an immune response, therefore for the treatment of a cancer. CAR modified y5 T cell or NK-cell-based immunotherapy provides improved methods for treating cancer and other diseases. In accordance, the present invention provide improved methods of immunotherapy to fine-tune the safety and efficacy of a cytotoxic response against target cells, e.g., tumor cells, expressing target antigens while decreasing the risk of on-target antigen, off- target cell cytotoxicity. The inherent migration tropism of y5 T cells or NK cells can increase T cells’ or NK cells’ targeting to cancer cells. In some embodiments, y5 T cells or NK-cells can act as a professional antigen presenting cells, combination therapies with other modalities of immunotherapy such as checkpoint inhibitors, oncolytic viruses, vaccines,ATTORNEY DOCKET NO. MIL-052WO1or cytokines could synergistically amplify recruitment and function of tumor-infiltrating lymphoid and non-lymphoid cells.

[0345] In some embodiments, cytokines, or armored CARs, compositions and CAR modified y5 T cells or NK cells may be used for the treatment of cancerous diseases, including tumorous diseases, including any malignancies that express the antigen. The administration of the composition(s) of the disclosure is useful for all stages (I, II, III, or IV) and types of cancer, including for minimal residual disease, early cancer, advanced cancer, and / or metastatic cancer and / or refractory cancer, for example. CAR and / or cytokine modified y5 T cells or NK-cells improve antitumor immune response. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the “cancer” or “cancer tissue” comprises a solid tumor.

[0346] In some embodiments, cancers for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0347] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinomaATTORNEY DOCKET NO. MIL-052WO1in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma;ATTORNEY DOCKET NO. MIL-052WO1neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease;; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; B-cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0348] As non-limiting examples, cytokines or CARs described herein, compositions and modified y5 T cells or NK cells are used for treating B cell related diseases / conditions. B cell related conditions include but are not limited to immunoregulatory conditions and hematological malignancies. In some embodiments, the disease is B cell malignancy, which refers to a type of cancer that forms in B cells (a type of immune system cell) as discussed infra. The B-cell related disease may be selected from the group consisting of plasmacytoma, Hodgkin lymphoma, follicular lymphoma, small non-cutting nuclear cell lymphoma, endemic Burkitt lymphoma, sporadic Burkitt lymphoma, marginal zone lymphoma, extranodal mucosal lymphoma Tissue lymphoma, nodular monocytic B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, mediastinal primary B cell lymphoma, lung B cell angiocentric lymphoma, Small lymphocytic lymphoma, B cell of unknown malignancy, lymphoma-like granulomatosis, post-transplant lymphoproliferative disorder, immunoregulatory disease, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiPhospholipid syndrome, Chagas disease, Graves’ disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, Multiple sclerosis, antiphospholipid syndrome, ANCA-related vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia, rapidly progressive glomerulonephritis, heavy chain disease, and primary or immune cell amyloidosis.ATTORNEY DOCKET NO. MIL-052WO1

[0349] In some embodiments, the cancer is a T-cell associated cancer. A T cell malignancy includes T-lymphoblastic lymphoma / leukemia, Cutaneous T-cell lymphomas (mycosis fungoides, Sezary syndrome, and others), Adult T-cell leukemia / lymphoma, Angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, nasal type, Enteropathy-associated intestinal T-cell lymphoma (EATL), Anaplastic large cell lymphoma (ALCL), and Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). An attenuated chimeric antigen receptor (CAR) comprising a T-cell antigen binding domain is disclosed for the treatment of various hematological malignancies including multiple myeloma. The T-cell antigen protein is expressed on a cancer cell. The antigen-binding portion of the CAR interacts with an epitope within the extracellular domain of the T-cell antigen fragment thereof.

[0350] In some embodiments, the present invention provides a method of inhibiting proliferation of or reducing a population of T-cell antigen-expressing cancer cells, the method comprising contacting a population of T-cell antigen -expressing cancer cells with modified y5 T-cells that express an attenuated CAR binding to the T cell antigen expressing cells. In certain aspects, the CAR and / or cytokine modified y5 T-cells or NK-cells reduce the number, amount, or percentage of cells and / or cancer cells in myeloid leukemia or another cancer associated with a T-cell antigen-expressing cell by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% relative to a negative control.

[0351] In some embodiments, a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of modified y5 T-cells or NK cells comprising one or more attenuated CARs. The genetically modified cells are a more efficacious and safer cellular immunotherapy by virtue of transducing a chemically regulatable immunostimulatory signal.

[0352] In various embodiments, the treatment results in a decrease in the prevalence, frequency, level, and / or amount of one or more symptoms or biomarkers associated with the disease being treated, e.g., a decrease of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of one or more symptoms or biomarkers as compared to a prior measurement in the subject or to a reference value.

[0353] The quantity and frequency of administration of modified immune effector cells will be determined by such factors as the condition of the patient, and the type and severity ofATTORNEY DOCKET NO. MIL-052WO1the patient's disease, although appropriate dosages and dose schedules may be determined by clinical trials.

[0354] One of ordinary skill in the art would recognize that multiple administrations of the compositions contemplated in particular embodiments may be required to effect the desired therapy. For example, a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more. Modified immune effector cells may be administered in the same or different compositions; in one or more compositions at the same time; or more than one composition at different times. Modified immune effector cells may be administered through the same route of administration or different routes.

[0355] The methods for administering the cell compositions contemplated herein include any method which is effective to result in reintroduction of ex vivo modified y5 T cells. One method comprises modifying blood derived y5 T cells or NK cells ex vivo by introducing one or more vectors encoding a cytokine and / or CAR and returning the transduced cells into the subject.EXAMPLES

[0356] These Examples are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to, limit its scope in any way. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.).Example 1: In vitro and in vivo Cell proliferation of engineered NK cells

[0357] This Example evaluated the anti-tumor activity, proliferation and in vitro tumorkilling activity of CB-NK cells engineered with IL-2 (H9, SEQ ID NO: 1).

[0358] CB-NK cells were engineered to express secreted IL2 (sIL2), secreted H9 (sH9), secreted IL-15 (sIL15), CD123-CAR-sIL15, un-transduced (UTD), or Target only using known methods in the art. Briefly, cryopreserved cord blood was thawed and rinsed. NK cells (CD3- / CD56+) were then isolated by negative selection using CD3 (T cells), CD19 (B Cells), and CD14 Microbeads. Enriched NK cells were stimulated with y-irradiated universal antigen presenting cells (uAPC). The NK cells were transfected with a retroviral vector encoding the cytokine / untransduced(UTD) / CD123-CAR-sIL15. The transduced CAR-NK cells were re-ATTORNEY DOCKET NO. MIL-052WO1stimulated with irradiated uAPC. Cultures were supplemented with IL-2 and cell density / viability is monitored throughout the expansion phase. To observe the in vitro effect of engineered NK cells with tumor cells, engineered CB NK cells were co-cultured with tumor cells (MV-4-11, Molml3, or KGla) following different effector-target (E: T) ratios (3:1 or 1: 1) for 2 weeks with Repeat Antigen Stimulation (RAS). During -2 weeks’ RAS assay, CB NK cells were rechallenged by fresh tumor cells as arrowhead indicated in FIG.2A-FIG. 2F. FIG. 2A shows reduction in tumor cell numbers with different cytokine engineering approaches in the absence of cytokine support, CB-NK engineered with sH9 demonstrated better tumor control in MV-4-11 at E: T ratio of 3: 1. FIG. 2B shows reduction in tumor cell numbers with different cytokine engineering approaches in the absence of cytokine support, CB-NK engineered with sH9 demonstrated better tumor control in Molml3 at E: T ratio of 3: 1. FIG. 2C shows NK-cell proliferation, CB-NK engineered with sH9 demonstrated higher cell numbers on a 2-weeks’ period co-culture with MV-4-11 at E: T ratio of 3: 1. FIG. 2D shows NK-cell proliferation, CB-NK engineered with sH9 demonstrated higher cell numbers on a 2-weeks’ period co-culture with Molml3 at E: T ratio of 3: 1.

[0359] On day 16, harvested engineered CB NK cells were evaluated in vivo by Molml3 model (CD123 antigen positive AML model). On day 3, 0.05 x 106Molml3 cells were inoculated into NSG mice.10 x 106fresh engineered CB NK cells were inoculated on day 0 (DO). Mice blood were sampled on days 6, 11 and 14 (D6, Dll, and D14). CK were analyzed on day 11 (Dll) by flow cytometry analysis and on D6, D14 by ddPCR assay. FIG. 2E is a histogram showing reduction in the number of tumor cells on DI 1, in CB-NK cells engineered with sH9, sIL2, and sIL15, more tumor cells were detected in no cytokine (UTD), or CD123-CAR-sIL15 groups as measured by flow cytometry. FIG. 2F shows that comparable or slightly more cell numbers were detected by flow cytometry in CB-NK cells engineered with sH9, compared to sIL15, better than CD123-CAR-sIL15. FIG. 2G shows CK analysis by ddPCR demonstrated comparable cell survival or proliferation in CB-NK cells engineered with sH9 and sIL15.

[0360] Overall, the results showed comparable or slightly better cell proliferation of H9 than IL-15 engineered CB-NK cells.Example 2: H9+IL2RP improves V81T product yield, proliferation and transduction efficiencyATTORNEY DOCKET NO. MIL-052WO1

[0361] This example analyzed the expansion of V51 T cells and the transduction efficiency of a MSLN CAR together with sH9, sH9T, sIL-2 or mbIL15 (IL-15 / IL-15Ra) in combination with IL2Rp.

[0362] Engineered blood-derived V51T cells were cultured for 14 days. During 14-day culturing process, V51 T cell proliferation was monitored by cell counting on D5 and D14. Fold of expansion was calculated from D5 to D14.

[0363] FIG. 3A shows fold of expansion of blood-derived V51T cells calculated from D5 to D14 of the MSLN CAR only; the MSLN CAR, membrane-bound IL-15 (mbIL15) and IL2RP co-expression; the MSLN CAR, secreted H9(sH9) and IL2RP co-expression (MSLN CAR_sH9+IL2RP); the MSLN CAR, secreted IL-2, and IL2RP co-expression; the MSLN CAR, secreted H9T, and IL2RP co-expression; Untransduced control. FIG. 3B shows transduction efficiency of the MSLN CAR. FIG. 3C shows gMFI of the MSLN CAR expression.

[0364] It was observed that MSLN CAR_sH9 + IL2RP group was comparable FoE to the MSLN CAR only, better than MSLN CAR-mbIL15 / Ra + IL2Rp. Additionally, V51 T cells transduced with a MSLN CAR together with sH9 and IL2RP demonstrated comparable or slightly better fold expansion and CAR expression during the V51 production process than the MSLN CAR only. In comparison, reduced fold expansion and CAR expression were observed in V51 T cells transduced with the MSLN CAR with membrane-bound IL15 (mbIL15) and IL2Rp.

[0365] To evaluate H9+IL2RP function, in vitro Repeat Antigen Restimulation (RAS) assay was assessed by both IncuCyte-based RAS assay and flow cytometry-based RAS assay. The MSLN CAR% was normalized to 40% for all groups. E: T ratio (3:1) was setup for both assays.

[0366] FIG. 3D shows results of IncuCyte-based RAS assay to evaluate sH9+IL2Rp function, in vitro RAS assay. FIG. 3E shows results of flow cytometry-based RAS assay. FIG. 3F shows results of flow cytometry performed to monitor V51 proliferation. It was observed that MSLN CAR_sH9 + IL2RP showed better proliferation during RAS assay.

[0367] To measure efficacy of sH9+IL2R|J in vivo, NSG mice were IP inoculated with 1 million GSU-luc cells on D3. On Day 0, mice were dosed with 5 x 106engineered V51 T cells. FIG. 3G shows bioluminescence (BLI) images taken on several timepoints as indicated. It was observed that MSLN CAR_sH9 + IL2RP shows better tumor control than the MSLN only group, and comparable efficacy to MSLN CAR-mbIL15 / Ra + IL2RP as the same as shown in FIG. 3H. FIG. 31 shows tumor control in cells treated with a PBS onlyATTORNEY DOCKET NO. MIL-052WO1control; the MSLN CAR only; the MSLN CAR, membrane-bound IL-15 (mbIL15) and IL2RP co-expression; the MSLN CAR, secreted H9 (sH9), and IL2RP co-expression; the MSLN CAR, secreted IL-2 (sIL2), and IL2RP co-expression; the MSLN CAR, secreted H9T (sH9T), and IL2RP co-expression. Only one mouse in MSLN CAR_sH9 + IL2RP group showed slightly less tumor control, which contributes into higher variation and less efficacy in Log scale BLI data.

[0368] CK analysis of blood-derived V51 T cells by ddPCR was performed to analyze proliferation.

[0369] Mice blood was collected for ddPCR CK analysis on D7 and D21. Without exogenous cytokine support, the MSLN CAR only shows limited V51 T cells expansion in vivo, which correlated with less efficacy in FIG. 3G and FIG. 3H. MSLN CAR_sH9 + IL2RP showed better cell expansion in late timepoint (FIG. 3J).

[0370] It was observed that V51T cells engineered with sH9 +IL2RP demonstrated improved proliferation.Example 3: Effect of Attenuated Membrane Bound IL-2 Muteins on GCC CAR expressing cell expansion, expression and function

[0371] This example demonstrates the effect of exemplary attenuated IL-2 muteins on cell expansion, GCC CAR expression, and maintenance of function.

[0372] FIG. 4A and FIG. 4B show a general schematic of the exemplary GCC CAR construct that was co-expressed with IL-2 muteins.

[0373] The results showed that in the absence of IL-2 Rp, there was less fold of expansion in cells co-expressing a CAR with a membrane-bound IL-2 attenuated mutein, while in the presence of IL-2RP, comparable fold expansion was observed (FIG. 5A) while maintaining high expression of the GCC CAR (FIG. 5B). Among the attenuated IL-2 muteins, AE88T and AE88R, which have the lowest binding affinities, were less impacted compared with the wild type or IL-2 muteins with higher affinities. This suggests that fine-tuning the binding affinity can mitigate the negative effects of cytokine engineering on cell production and CAR expression. Further, upon repeated antigen stimulation with HT29-GCC (without IL-2RP), it was noted that expression of membrane-bound IL-2 attenuated muteins (AE88T and AE88R) led to a significant decrease in tumor cell count relative to just GCC CAR alone (FIG. 5C).Further, upon repeated antigen stimulation with HT29-GCC, the proliferation of V51 (VD1) cells was lesser by day 13, noting that CARs co-expressing membrane-bound IL-2 AE88T induced relatively high proliferation of V51 cells (FIG. 5D). It was also observed that co-ATTORNEY DOCKET NO. MIL-052WO1expression of attenuated IL-2 mutein had a decrease in STAT5 activation in a standard transpresentation reporter cell line assay known in the art (FIG. 5E).

[0374] Similar results were observed in GCC CAR expressing cells that co-expressed membrane-bound IL-2 molecules, wherein the fold expansion was lesser (FIG. 6A) than the GCC CAR alone, but a high expression of GCC CAR was maintained (FIG. 6B). Moreover, the quantification of results shown in FIG. 5C further demonstrated the ability of the membrane-bound IL-2 attenuated CAR to target HT29 / GCC antigen (FIG. 6C). It was further observed that there was a decrease in V51 proliferation of all GCC CAR constructs co-expressing membrane-bound IL-2, except for GCC CAR attenuated IL-2 mutein AE88T (FIG. 6D)

[0375] Overall, the results from this example showed that expansion, GCC CAR expression and function were comparable or higher in the presence of attenuated membrane bound IL-2.Example 4: Effect of Attenuated membrane bound IL-2 Muteins on Signaling, Proliferation, Effector Function, CAR Maintenance, and Tumor Killing

[0376] This example shows the effects of expression of exemplary attenuated IL-2 muteins on expression of armoring, CAR maintenance, and other functional parameters.

[0377] To assess the signaling capabilities of attenuated IL-2 muteins, HEK-Blue IL-2 / IL-15 STAT5 reporter cells were transduced with either wild-type IL-2 or attenuated IL-2 muteins, which were either secreted (sIL2) or membrane-bound (mbIL2). The activation of the JAK / STAT5 signaling pathway was assessed by the evaluation of STAT5-SEAP reporter activity detection. As compared to the wild-type IL-2 in both forms, the IL-2 muteins exhibited a gradual and affinity-dependent reduction in JAK / STAT5 signaling, with IL2AE88R showing a highly reduced signal (FIG. 7A).

[0378] Attenuating mbIL2 affinity stabilizes armoring expression with minimal transpresentation and enhances potency

[0379] To assess attenuated mb IL-2 muteins function, mbIL-2 muteins were co-expressed with GCC CAR in V51 T cells. Lower affinity mbIL-2 muteins stabilized IL-2 expression on V51 T cell surface in FIG. 7B (top). When co-cultured engineered V51 T with HEK-Blue IL-2 / IL-15 STAT5 reporter cells, lower affinity mb IL-2 muteins exhibits lower transpresentation in FIG. 7B (bottom). Lower affinity mbIL-2 muteins (mbIL-2-AE88T and mbIL-2-AE88R) maintained V51 potency (FIG. 7C top) and proliferation (FIG. 7C bottom) when engaged with tumor cells (LSI 034) during in vitro Repeat Antigen RestimulationATTORNEY DOCKET NO. MIL-052WO1(RAS) assay, assessed by both IncuCyte-based RAS assay (FIG. 7C top) and flow cytometrybased RAS assay (FIG. 7C bottom).

[0380] Similarly, the membrane-bound IL-2 muteins comprising a Q126T mutation and / or mutations that bias it towards reduced binding to IL-2Ra (z.e., F42A and R38E) versus that do not exhibit such bias were found to attenuate the JAK / STAT5 signaling in a similar fashion, with muteins comprising the N88R / N88E / N88T and Q126T mutations showing greater reduction in reporter activity (FIG. 7D). However, transduction efficiency of the IL-2 mutein armoring or CAR into the HEK-Blue IL-2 / IL-15 cells was unaffected (FIG. 7E).Example 5: Effect of Signal Peptide Expression on Attenuated mbIL-2 mutein Armored CAR Cellular Parameters and Function

[0381] This example demonstrates the impact of the presence of a signal peptide on exemplary mbIL-2 muteins on armoring and CAR expression and killing activity.

[0382] The signal peptide of a membrane-bound protein, such as the IL-2 mutein contemplated herein, is a short amino acid sequence on nascent proteins that directs a protein to the endoplasmic reticulum of a cell, thus labeling it for secretion or insertion into a membrane. Without wishing to be bound by theory, signal peptides are typically between 20 to 40 amino acids in length.

[0383] The effects of anti-GCC CARs expressing mbIL-2 muteins with signal peptides, particularly long signal peptides (LSP; SEQ ID NO: 74), on detection of cells expressing IL-2 or GCC CARs (unarmored or armored; mbIL2AE88T or mbIL2AE88R) and potency across two (n=2) donors.

[0384] In expressing GCC CARs with mbIL2 muteins that contain LSPs, a notable reduction in the average expression of IL-2 was observed, while the presence of the LSP exhibited limited impact on CAR expression (FIGs. 8A-8D). FIGs. 8E-8H shows RAS assays against GCC expressing tumor cells (FIGs. 8E and 8G, respectively) and V51 proliferation at the RAS endpoint (FIGs. 8F and 8G, respectively) across donors. While GCC CARs expressing the mbIL2 armoring showed greater potency than unarmored GCC CAR cells or untransduced cells, armored CARs expressing the LSP on the mbILAE88T showed similar killing activity and proliferation to those without it. However, the GCC CAR expressing the mbIL2AE88R with the LSP showed slightly reduced killing activity, but similar V51 proliferation as other CAR constructs.

[0385] This, in turn, suggests that not only the specific combination of attenuated mbIL2 mutations can affect in vitro and in vivo activity of CAR cells, but that the activity andATTORNEY DOCKET NO. MIL-052WO1expression of the CAR can further be regulated by the presence of a signal peptide on the mbIL2 armoring.Example 6: Effect of IL-2 for armoring over unarmored CAR in vivo

[0386] This example tested in vivo anti-tumor of GCC CAR cells with attenuated IL-2 muteins.

[0387] Briefly, NSG were inoculated SQ with LS1034 cells and treated with a dose of 4.5×106CAR+cells normalized to 50% CAR+using cells from n=2 donors, and tumor volume was tracked over the course of 28 days. It was observed that GCC CAR y5 T cells armored with mbIL2AE88R showed single agent activity and improved killing in comparison to unarmored GCC CAR cells and a PBS control (FIG. 9).Example 7: Improved cytotoxicity and proliferation of_CD19 CAR-ydT cells armored with membrane-bound attenuated IL-2 muteins

[0388] This example further tested effect of membrane bound attenuated IL-2 muteins on CD 19 CAR expressing y T cells.

[0389] IL-2 muteins were generated without mutations that do bias the mutein towards binding IL-2Ra (e.g., without the F42A and R38E mutations; “non-a-biased”), and incorporation of a mutation with disruption of binding at IL-2R. P (z.e., Q126T), to compare their impact on CAR effector function and anti-tumor activity. FIGs. 10A-10C show data for anti-CD19 CAR y5 T cells armored with attenuated IL-2 muteins that are either a-biased or not a-biased on tumor killing in vitro. It was observed that CAR y5 T cells armored with attenuated IL-2 muteins that are not a-biased show enhanced potency in comparison to armored-CAR cells expressing the IL-2 muteins that are a-biased. It was also noted that a-biased muteins comprising amino acid substitutions of N88R or N88T mutations exhibited better potency, while N88E showed reduced potency in comparison, but still better than the unarmored CD 19 CAR.

[0390] Moreover, anti-CD19 CAR y5 T cells armored with attenuated IL-2 muteins, regardless of a-bias, exhibited better y5 cell proliferation and CAR persistence than without armoring (FIGs. 10D-10G).

[0391] CAR-T cells expressing membrane-bound, attenuated IL-2 muteins enhance tumor targeting with a favorable safety profile. Fine-tuning IL-2 binding affinity — toward a-bias,ATTORNEY DOCKET NO. MIL-052WO1non-a-bias, or reduced y-chain binding — offers design flexibility across CAR and TCR platforms.EQUIVALENTS AND SCOPE

[0392] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims:

Claims

ATTORNEY DOCKET NO. MIL-052WO1CLAIMS1. An immune cell expressing a recombinant IL-2 mutein, wherein the immune cell is a blood derived natural killer (NK) cell or a y5 T cell, and wherein the IL-2 mutein has reduced binding to IL2 receptor (IL2R).

2. The immune cell of claim 1 wherein the recombinant IL-2 mutein has reduced binding to IL2Rp.

3. The immune cell of claim 1 or 2, wherein the recombinant IL-2 mutein comprises one or more amino acid substitutions selected from the group consisting of L18R, Q22E, R38E, F42A, L80F, R81D, L85V, I86V, N88R, N88T, V91T, I92F, Q126A, Q126C, Q126E, Q126H, Q126K, and Q126T, numbered in accordance with wild-type human IL-2.

4. The immune cell of claim 2, wherein the recombinant IL-2 mutein comprises the amino acid substitutions ofa) L80F, R81D, L85V, I86V, and I92F;b) L80F, R81D, L85V, I86V, I92F, and Q126T;c)L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126H;d) L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126K;e) L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126A;f) L80F, R81D, L85V, I86V, I92F, L18R, Q22E, Q126C;g)F42A, R38E;h)F42A, R38E, V91T;i) F42A, R38E, Q126E;j) F42A, R38E, N88T; ork) F42A, R38E, N88R5. The immune cell of any one of claims 1-3, wherein the recombinant IL-2 mutein comprises a sequence having 70% or greater amino acid sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 11.ATTORNEY DOCKET NO. MIL-052WO16. The immune cell of any one of claims 1-3, wherein the recombinant IL-2 mutein comprises a sequence having 80%, 85%, 90%, 95% or greater amino acid sequence identity to any one of SEQ ID NO: 1 to SEQ ID NO: 11.

7. The immune cell of any one of claim 1-3, wherein the recombinant IL-2 mutein comprises any one of SEQ ID NO: 1 to SEQ ID NO: 11.

8. The immune cell of any one of claims 1-7, wherein the recombinant IL-2 mutein is a soluble form of the recombinant IL-2 mutein.

9. The immune cell of any one of claims 1-7, wherein the recombinant IL-2 mutein is a membrane bound form of the recombinant IL-2 mutein.

10. The immune cell of any one of claims 1-9, wherein the recombinant IL-2 mutein further comprises a signal peptide.

11. The immune cell of claim 10, wherein the signal peptide comprises the sequence of MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA (SEQ ID NO: 74).

12. The immune cell of any one of claims 9-11, wherein the membrane bound form of the recombinant IL-2 mutein comprises a hinge and transmembrane domain selecting from CD4, CD8, CD28, orB7.

13. The immune cell of claim 12, wherein the membrane bound form of the recombinant IL-2 mutein comprises a hinge and transmembrane domain derived from CD8.

14. The immune cell of any one of claims 8-13, wherein the membrane bound form of the recombinant IL-2 mutein further comprises a CTLA4 intracellular domain.

15. The immune cell of any one of claims 1-14, wherein the immune cell further expresses a chimeric antigen receptor (CAR).

16. The immune cell of claim 15, wherein the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and at least one intracellular signaling domain.

17. The immune cell of claim 16, wherein the extracellular antigen binding domain is an IgA antibody, IgG antibody, IgE antibody, IgM antibody, bi- or multi- specific antibody, Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fd’ fragment, Fd fragment, isolated CDRs or sets thereof; single-chain variable fragment (scFv), polypeptide-Fc fusion, single domain antibody (sdAb), camelid antibody; masked antibody, Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain, Tandem diabody, VHHs, Anticalin, Nanobody, humabody, minibodies, BiTE, ankyrin repeat protein, DARPIN, Avimer, DART,ATTORNEY DOCKET NO. MIL-052WO1TCR-like antibody, Adnectin, Affilin, Trans-body; Affibody, TrimerX, MicroProtein, Fynomer, Centyrin; and KALBITOR; optionally wherein the GCC binding agent is a single domain antibody (sdAb) or a heavy chain only antibody, or fragment thereof.

18. The immune cell of claim 16 or 17, wherein the extracellular antigen domain binds a tumor associated antigen selected from 4- IBB, 5 AC, 5T4, A2aR, activin receptor-like kinase 1, AGS-22M6, AKAP4, alpha-fetoprotein, angiopoietin 2, B7-H3, BAFF, BAGE, BCR-ABL, BORIS, CA-125, CA19-9, C242 antigen, carbonic anhydrase 9 (CA-IX), CCR4, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD28, CD30 (TNFRSF8), CD33, CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 v6, CD51, CD56, CD70, CD71, CD73, CD74, CD79B, CD80, CD137, CD140a, CD152, CD200, CD221, CD274, CEA, ch4D5, CLDN18.2, CS1, CSF1R, CTLA-4, C-X-C chemokine receptor type 4, DLL4, DR5, EBAG9, EGF, EGFR, EGFL7, EpCAM, ERBB2, ERBB3, FAP, fibronectin extra domain-B, folate receptor 1, folate receptor alpha, folate hydrolase, Frizzled receptor, GAGE, GD2 ganglioside, GD3 ganglioside, glioma, glypican 3, GP MB, gp100, guanylate cyclase 2C (GUCY2C), HER1, HER2 / neu, HER3, HGF, HHGFR, histone complex, HLA-DR, human scatter factor receptor kinase, HPV-16, HSP105, IDH1, IDO1, IGF-I, IGF-1 receptor, ILGF2, IL-6, IL-13, integrin avP3, integrin a5pi, KIR, LAG-3, Lewis-Y antigen, LY6K, MAGE-1, MAGE- A3, MAGE-C2, MAGE-D4, MAPG, MART-1, Melan-A, MET, MCP-1, mesothelin, MIF, MSLN (Mesothelin), MS4A1, mucin CanAg, MUC1, MUC4, MUC16, NG2, N-glycolylneuraminic acid, Notch receptor PD-1, NY-ESO-1, OCAA, PAP, PDGF-Ra, PDCD1, PD1, PD-L1, phosphate-sodium co-transporter, phosphatidylserine, PRAME, PSA, RANKL, RON, R0R1, SDC1, Sialyl-Tn, SLAMF7, SPAG-9, SSX1, STEAP1, survivin, TAG- 72, telomerase, TEM1, tenascin C, TGF-P, TFM-3, TLR, TAM, TFM-3, TRAIL-R2, TRAIL-R1, TWEAK receptor, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor antigen CTAA16.88, TYRP1 (glycoprotein 75), VEGF-A, VEGFR2, VEGFR-1, vimentin, VISTA, WT1, and XAGE-lb.

19. The immune cell of claim 18, wherein the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) binding domain that binds GUCY2C.

20. The immune cell of claim 19, wherein the extracellular antigen binding domain is an anti -guanyl ate cyclase 2C (GUCY2C) binding domain that binds GUCY2C and comprises:ATTORNEY DOCKET NO. MIL-052WO1a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of HYYWS (HCDR1; SEQ ID NO: 21), RIYPSGSTSYNPSLKS (HCDR2; SEQ ID NO: 26), and DRSTGWSEWNSDL (HCDR3; SEQ ID NO: 31);a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMS (HCDR1; SEQ ID NO: 24), KIRHDGGEKYYVDSVKG (HCDR2; SEQ ID NO: 27), and DYTRDV (HCDR3; SEQ ID NO: 32);a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIKYDGSEKYYADSVKG (HCDR2; SEQ ID NO: 28), and DYNKDY (HCDR3; SEQ ID NO: 33);a heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIRHDGGEKYYPDSVKG (HCDR2; SEQ ID NO: 29), and DYNKDL (HCDR3; SEQ ID NO: 34); ora heavy chain variable region (VH) with complementarity determining region (CDR) sequences of RYWMT (HCDR1; SEQ ID NO: 25), KIRHDGGEKYYADSVKG (HCDR2; SEQ ID NO: 30), and DYNKDY (HCDR3; SEQ ID NO: 33).

21. The immune cell of claim 20, wherein the anti- guanylate cyclase 2C (GUCY2C) binding domain comprises a VH sequence in Table 2 or Table 3.

22. The immune cell of claim 20 or 21, wherein the CAR further comprises a CD28 hinge domain, a CD28 transmembrane domain, a 4- IBB co-stimulation domain and a CD3z activation domain.

23. The immune cell of claim 18, wherein the extracellular antigen binding domain binds to CD 19.

24. The immune cell of any one of claims 1-23, wherein the immune cell further expresses an IL-2RaPY.

25. The immune cell of any one of claims 1-24, wherein the immune cell further expresses a recombinant IL-2Rp.ATTORNEY DOCKET NO. MIL-052WO126. The immune cell of any one of the preceding claims, wherein the immune cell comprises a switch receptor comprising Fas and / or 0X40.

27. The immune cell of claim 26, wherein the immune cell further expresses a polypeptide comprising a dominant-negative Fas (Fas-DN), a Fas-CD27 chimeric polypeptide (Fas-CD27), a Fas-4-lBB chimeric polypeptide (Fas-BB), a Fas-OX40 chimeric polypeptide (Fas-OX40), or any combination thereof.

28. The immune cell of any one of the preceding claims, wherein NKp30 and CD56 expression is elevated.

29. The immune cell of any one of the preceding claims, wherein the immune cell comprises greater cytotoxicity, potency, proliferation and / or anti-tumor activity relative to a control immune cell that does not express an IL-2 mutein.

30. The immune cell of any one of the preceding claims, wherein the IL-2 mutein is expressed under an inducible promoter.

31. The immune cell of claim 30, wherein the inducible promoter is an NF AT minimal promoter.

32. The immune cell of any one of claims 1-29, wherein the IL-2 mutein is expressed under a constitutive promoter.

33. The immune cell of claim 32, wherein the promoter is 5' LTR or EFS in SIN vector.

34. The immune cell of any one of claims 1-33, wherein the immune cell does not express a recombinant IL- 13 superkine.

35. The immune cell of any one of the preceding claims, wherein the immune cell is a cord-blood derived natural killer (CB-NK) cell.

36. The immune cell of any one of claims 1-34, wherein the immune cell is a y5 T cell.

37. The immune cell of claim 36, wherein the y5 T cell is isolated / derived from a tissue (e.g., either a hematopoietic or non-hematopoietic tissue sample) or derived from a pluripotent stem cell.

38. The immune cell of claim 37, wherein the y5 T cell is isolated / derived from the blood or the skin.

39. The immune cell of any one of claims 36-38, wherein the y5 T cell is a Vdeltal cell.ATTORNEY DOCKET NO. MIL-052WO140. The immune cell of any one of the preceding claims, wherein the immune cell is allogeneic.

41. The immune cell of any one of the preceding claims, wherein the immune cell is expanded.

42. A polynucleotide encoding the IL-2 mutein of any one of claims 1-14 and 30-34.

43. A composition comprising a population of the immune cells of any one of claims 1-41.

44. A method of modulating immune function comprising administering to a patient in need thereof an immune cell of any one of claims 1-41 or the composition of claim 43.

45. The method of claim 44, wherein administration of the immune cell leads to increased proliferation of immune cells, cytotoxicity, potency and / or tumor killing.

46. A method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of an immune cell of any one of claims 1-41 or the composition of claim 43.

47. The method of claim 46, wherein the cancer is selected from acute myeloid leukemia, gastrointestinal cancer, colorectal cancer, colorectal adenocarcinoma, colorectal leiomyosarcoma, colorectal lymphoma, colorectal melanoma, a colorectal neuroendocrine tumor, metastatic colon cancer, stomach cancer, gastric adenocarcinoma, gastric lymphoma, gastric sarcoma, esophageal cancer, squamous cell carcinoma, adenocarcinoma of the esophagus, or pancreatic cancer.

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