Single-and multi-cytokine fusion proteins and uses thereof

Abstract

Described herein are single- and multi-cytokine fusion proteins as well as cytokine VHH fusion proteins. Method of producing these fusion proteins as well as their uses are also described. Further included are method of using the fusion proteins as separate compositions that are administered to a subject in need thereof.

Description

SINGLE- AND MULTI-CYTOKINE FUSION PROTEINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent applications No. 63 / 472,939 filed June 14. 2023. No. 63 / 472.972. filed June 14. 2023. No. 63 / 527.948. filed July 20,2023, No. 63 / 530,784, filed August 4, 2023, and No. 63 / 641,265, filed May 1, 2024, tire entirety of each is hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable fonn named ri)96034_000006WOPT_SequcnccListing.xml. having a size in bytes of 1,254,546 bytes, and created on June 13.2024. The information contained in this computer readable form is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0003] This invention relates to fusion proteins and their uses in treating disease conditions such as cancer.SUMMARY OF THE INVENTION

[0004] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0005] Various embodiments provide for a fusion protein comprising a scaffold polypeptide and at least two of(a)-(c): a. one or more of an interleukin 15 (IL- 15) variant, b. one or more of an interleukin 18 (IL-18), a fragment of the IL-18, an IL-18 variant, or a fragment of the IL- 18 variant, and c. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA).

[0006] In various embodiments, the one or more polypeptides capable of binding the TAA can comprise a receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH.

[0007] In various embodiments, tire TAA can comprise EGFR, HER2, or DLL.

[0008] In various embodiments, the activation receptor can comprise cluster of differentiation (CD) 3, CD 16, 9 TCR, δ2TCR or 81 TCR, or wherein the costimulatory receptor can comprise cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40.

[0009] In various embodiments, the fusion protein can comprise one or more of an interleukin 15 (IL-15) variant and one or more of an interleukin 18 (IL-18), a fragment of the IL-18, an IL-18 variant. In various embodiments, the fusion protein can comprise one or more of IL- 15 variants and one or more IL- 18 variants.

[0010] In various embodiments, the fusion protein can further comprise a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD 16,TCR, 52 TCR or 51 TCR, and wherein optionally the costimulatoiy receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40.

[0011] In various embodiments, wherein the IL- 15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18, the IL-18 variant, orthe fragment of the IL-18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH, can be each independendy fused to the C-terminus of the scaffold polypeptide. In various embodiments, the IL-15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, can be each independendy fused to the N-tenninus of the scaffold polypeptide. In various embodiments, the scaffold polypeptide is an antibody, the IL- 15 variant, the interleukin 18 (IL-18), the fragment of the IL-18, the IL-18 variant, or the fragment of the IL-18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, can be each independently fused to any one of one of a C-terminus or an N-tenninus of a heavy chain or a light chain, or fused to any? one of a CH2 domain, or hinge region of the antibody.

[0012] In various embodiments, the scaffold polypeptide can be an antibody or a fragment thereof. In various embodiments, the antibody can be an IgA, IgM, IgG, or IgE antibody. In various embodiments, the antibody can be an anti-PD-1 antibody or anti-PD-Ll antibody.

[0013] In various embodiments, the scaffold polypeptide can be a fragment cry stallizablc (Fc) region or a fragment tiiereof.In various embodiments, the fragment cry stallizablc (Fc) region can be an Fc region from an IgG4, knobs-in-hole (KiH) Fc, or IgGl. In various embodiments, the fragment crystallizable (Fc) region can be a knobs-in-hole (KiH) Fc.

[0014] In various embodiments, the scaffold polypeptide can be a polypeptide or protein capable of translocating into an endoplasmic reticulum (ER), or a fragment thereof.

[0015] In various embodiments, the scaffold polypeptide can be selected from fragment cry stallizablc (Fc) region, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin. designed ankyrin repeat protein (DARPin), a binding domain thereof. and a fragment thereof.

[0016] In various embodiments, IL-15 variant can comprise the sequence of Formula I:XiWVX4VISDLKKIEDLIQSMHIX22ATLYTESX3oVHPSCKVTAMX iCFLX45ELQX49lSLX53SGDASIHDTVXc4N LX67X68LANNSLSSNGX79VTESGCKECEELEX93KNIKEFLQSX103VHIVX108MFIX112TS, wherein a. Xi is any amino acid,b. X4 is any amino acid, c. X22 is any amino acid, d. X30 is any amino acid, e. X41 is any amino acid, f. X45 is any amino acid, g. X49 is any amino acid except V, h. X53 is any amino acid except E, i. Xe4 is any amino acid, j . X„7 is any amino acid, k. Xg8 is any amino acid, l. X79 is any amino acid, m. X93 is any amino acid, n. X103 is any amino acid, o. Xios is any amino acid, and p. Xi 12 is any amino acid; (SEQ ID NO:533).

[0017] In various embodiments, tire IL- 18 variant can comprise al. amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFnSMYKDSQPRGMAVTISVKVEKISTLSVENKnSFKEMNPPDNIKDTKSDIIFFQRSVPGHDN KMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250), with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250, OR a2. amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLHDQGNRPLFEDMTDS DCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDN KMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251, optionally further comprising amino acid substitutions at one or more of C74, C104, Cl 12, and C164. each independently substituted to valine, alanine or serine, OR a3. an IL-18 variant selected from Table 6B; and bl. optionally, wherein the one to five amino acid substitutions are one or more of: E42K, E42R, E42A, E42H, or E42Q; M87K, or M87H; K89G, K89A, or K89E; M96L, or M96I; or M149V or M149I; or b2. optionally, wherein the one to five amino acid substitutions are E42K, E42R, E42A, E42H, or E42Q; M87K, orM87H; K89G, K89A, or K89E; M96L. or M96I; and M149V or M149I.

[0018] In various embodiments, the IL- 18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL- 18 variant further can comprise its propeptide (PP) or a PP variant, and optionally, wherein the PP or the PP variant is on the N-tcrminus end relative to the IL- 18, the fragment of IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant.

[0019] In various embodiments, the IL-18, tire fragment of IL-18, the IL-18 variant, or the fragment ofthe IL- 18 variant can further comprise a short polypeptide or protein.

[0020] In various embodiments, the fusion protein can further comprise one or more cleavage sites and the fusion protein is cleaved at the one or more cleavage sites by one or more proteases, hr various embodiments, the one or more cleavage sites can be between the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant and tire scaffold polypeptide, or within the PP, between PP or the PP variant and the IL- 18, a fragment of IL- 18, an IL- 18 variant, or a fragment of the IL- 18 variant, or within the PP, between the PP or the PP variant and the scaffold polypeptide, or within the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment ofthe IL-18 variant, or within the PP, or a combination thereof.

[0021] In various embodiments, the ROR1 VHH can comprise: a polypeptide having SEQ ID NO:325 (complementarity-detennining region (CDR) 1 of 2A11), a polypeptide having SEQ ID NO:326 (CDR2 of 2A11). a polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination thereof; OR a variant of the polypeptide having SEQ ID NO:325 (CDR1 of 2A11), a variant of the polypeptide having SEQ ID NO:326 (CDR2 of 2A11), a variant of the polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination thereof, wherein the variant of the polypeptide having SEQ ID NO: 325 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:325, wherein the variant of the polypeptide having SEQ ID NO:326 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:326, wherein the variant ofthe polypeptide having SEQ ID NO:327 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:327, and wherein: the variant of the polypeptide having SEQ ID NO:325 and the variant of the polypeptide having SEQ ID NO:327 do not replace cysteine residues in the polypeptide having SEQ ID NO:325 and the polypeptide having SEQ ID NO:327. or the variant of the polypeptide having SEQ ID NO:325 and the variant of the polypeptide having SEQ ID NO:327 replaces one or both of the cysteine residues in the polypeptide having SEQ ID NO:325 and / or one or both of the cysteine residues in the polypeptide having SEQ ID NO:327 with an amino acid that contains a cross-linking functional group.

[0022] Various embodiments provide for a polynucleotide encoding any one of the fusion proteins of the invention described above. Various embodiments provide for a polynucleotide encoding any one of the fusion proteins of the invention described herein.

[0023] Various embodiments provide for an expression vector comprising any one of the polynucleotides of the invention described above. Various embodiments provide for an expression vector comprising any one of the polynucleotides of the invention described herein.

[0024] Various embodiments provide for a cell transfected with any one of the expression vectors of the invention described above. Various embodiments provide for a cell transfected with any one of the expression vectors of the invention described herein. In various embodiments, the cell can be a mammalian cell. In various embodiments, the mammalian cell can be a CHO cell or a HEK-293 cell. In various embodiments, the cell can be a bacterial cell or yeast cell.

[0025] Various embodiments provide for a method of producing any one of the fusion proteins described above, comprising: culturing any one of the cells described above, in cell culture medium to allow the fusion protein to be produced, and optionally secreted into the cell culture medium. Various embodiments provide for a method of producing any one of the fusion proteins described herein, comprising: culturing any one of the cells described herein, in cell culture medium to allow the fusion protein to be produced, and optionally secreted into the cell culture medium. In various embodiments, the method can further comprise isolating the fusion protein, In various embodiments, the method can further comprise purifying the fusion protein.

[0026] Various embodiments of the invention provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering any one of the fusion proteins described above to a subject in need thereof. Various embodiments of the invention provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering any one of the fusion proteins described herein to a subject in need thereof. In various embodiments, the subject has cancer.

[0027] Various embodiments provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need tiiereof, comprising administering any one of the fusion proteins described above to a subject in need dicrcof. Various embodiments provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering any one of the fusion proteins described herein to a subject in need thereof. In various embodiments, the disease or condition can be cancer.

[0028] Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering at least two of the following to a subject in need thereof: a. interleukin 15 (IL- 15) variant or IL- 15 fusion protein, b. interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, a fiagment of the IL-18 variant, or an IL-18 fusion protein, c. a multispecific antibody construct comprising i. a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, andii. a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally tire activation receptor comprises cluster of differentiation (CD) 3, CD 16, v9 TCR, 82 TCR or 81 TCR and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40, and d. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA).

[0029] In various embodiments, tire subject has cancer.

[0030] Various embodiments provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering at least two of the following to a subject in need thereof: a. interleukin 15 (IL- 15) variant or IL- 15 fusion protein, b. interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, a fragment of tire IL-18 variant, or an IL-18 fusion protein, c. a multispecific antibody construct comprising iii . a receptor tyrosine kinase-like orphan receptor 1 (R0R1 ) VHH, and iv. a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD 16, v9 TCR, 82 TCR or 81 TCR, and w herein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40, and d. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA).

[0031] In various embodiments, the disease or condition is cancer.

[0032] Various embodiments provide for an interleukin 15 (IL-15) variant comprising the sequence ofFormula I:X1WVX4VISDLKKIEDLIQSMHIX22ATLYTESX30VHPSCKVTAMX41CFLX45ELQX49ISLX53SGDASIHDTVX64NLX67X68LANNSLSSNGX79VTESGCKECEELEX93KNIKEFLQSX1O3VHIVXW8MFIX112TS, wherein a. Xi is any amino acid. b. X4 is any amino acid, c. X22 is any amino acid, d. X30 is any ammo acid, e. X41 is any amino acid,

[0036] In various embodiments, X49can be R or K, and X53can be G, K, I or A; (SEQ ID NO:564).In various embodiments, X30can be N (SEQ ID NO:565). In various embodiments,can be Q (SEQ ID NO:566). In various embodiments, Xi can be G (SEQ ID NO:567).

[0037] In various embodiments, the IL-15 variant can comprise an IL-15 variant listed in Table 1A or Table 3A.

[0038] Various embodiments provide for an IL-15 fusion protein, comprising any one of the IL-15 variants described above; and a scaffold polypeptide. Various embodiments provide for an IL-15 fusion protein, comprising any one of the IL- 15 variants described herein; and a scaffold polypeptide.

[0039] In various embodiments, the IL- 15 variant can be fused to the C-tenninus of the scaffold polypeptide.

[0040] In various embodiments, the scaffold polypeptide can be an antibody or a fragment thereof. In various embodiments, the antibody can be an IgA, IgM, IgG, or IgE antibody. In various embodiments, the antibody can be an anti-PD-1 antibody or anti-PD-Ll antibody.

[0041] In various embodiments, the scaffold polypeptide can be a Fc region or a fiagment thereof. In various embodiments, the scaffold polypeptide can be a Fc region or a fiagment thereof and the scaffold polypeptide does not comprise an Fab. In various embodiments, the Fc region can be an Fc region from an IgG4, knobs-in-hole (KiH) Fc, or IgGl. In various embodiments, the Fc region can be a knobs-in-hole (KiH) Fc. In various embodiments, the IL-15 variant can be fused to the knob of the KiH Fc. In various embodiments, the IL-15 variant can be fused to the hole of the KiH Fc.

[0042] Various embodiments provide for a polynucleotide encoding any one of the IL-15 variants described above, or any one of the 11-15 fusion proteins described above. Various embodiments provide for a polynucleotide encoding any one of the IL- 15 variants described herein, or any one of the 11-15 fusion proteins described herein.

[0043] Various embodiments provide for an expression vector comprising any one of the polynucleotides of encoding any one of the IL- 15 variants described above, or any one of the 11-15 fusion proteins described above. Various embodiments provide for an expression vector comprising any one of the polynucleotides of encoding any one of the IL- 15 variants described herein, or any one of the IL- 15 fusion proteins described herein.

[0044] Various embodiments provide for a cell transfected with any one of the expression vector comprising any one of the polynucleotides of encoding any one of the IL- 15 variants described above, or any one of the 11-15 fusion proteins described above. Various embodiments provide for a cell transfected w ith any one of the expression vector comprising any one of the polynucleotides of encoding any one of the IL- 15 variants described herein, or any one of the 11-15 fusion proteins described herein. In various embodiments, the cell can be a mammalian cell, In various embodiments, the mammalian cell is a CHO cell or a HEK-293 cell. In various embodiments, the cell can be a bacterial cell or yeast cell.

[0045] Various embodiments provide for a method of producing any one of the IL- 15 variants or any one of the IL- 15 fusion proteins described above, comprising: culturing any one of the cells described above, in cell culture medium to allow the IL- 15 variant or the fusion protein to be produced, and optionally secreted into the cell culture medium. Various embodiments provide for a method of producing any one of the IL-15 variants or any one of the IL-15 fusion proteins described herein, comprising: culturing any one of the cells described herein, in cell culture medium to allow the IL-15 variant or the fusion protein to be produced, and optionally? secreted into the cell culture medium. In various embodiments, the method can further comprise isolating the IL- 15 variant or the fusion protein, or purifying the IL- 15 variant or the fusion protein.

[0046] Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering any of the IL- 15 variants described above, or any one of the IL- 15 fusion protein described above to a subject in need thereof Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering any of the IL- 15 variants described herein, or any one of the IL- 15 fusion protein described herein to a subject in need thereof. In various embodiments, the subject has cancer.

[0047] Various embodiments provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering any of the IL-15 variants described above, or any one of the IL-15 fusion protein described above to a subject in need tiiereof. Various embodiments provide for a mediod of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering any of the IL-15 variants described herein, or any one of the IL- 15 fusion protein described herein to a subject in need thereof. In various embodiments, the subject has cancer.

[0048] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0049] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative ratherdian restrictive.

[0050] Figures 1A-1D depict nonlimiting examples of the fusion proteins in accordance with various embodiments of the present invention.

[0051] Figures 2 A-2D show the impact of an exemplary anti-PD 1 -pro-IL- 18 antibody, one of three exemplary anti-PD 1 -mutant-IL- 15 antibodies or the combination of the anti-PD 1 -pro-IL- 18 antibody and of each of the three mutant IL- 15 antibody fusions on IFNy release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (Pelicluster CD3, which is capable of crosslinking CD3 and activating T cells independent of FcyR binding) for 72 hours. For anti-PD 1 -pro-IL- 18, the exemplar}? test article was Fuse694, which incorporates one pro-IL- 18 (granzyme B cleavable IL18mutAS) on the C-terminal knob or hole of an IgGl-LALA version of nivolumab (Fuse691). For anti- PDl-mutant-IL-15, tire three test articles were Fuse765 (FIG. 2A, open triangle, dashed line), Fuse773 (FIG. 2B. open triangle, dashed line) and Fuse774 (FIG. 2C, open triangle, dashed line), which respectively incorporated one IL-15m4, IL15m9 or IL 15m 10 on the C-temtinal knob or hole of Fuse691. FIGs 2A-2C are non-linear x-y plots of test article concentration versus IFNy release. A summary table of EC50, Emax and AUC (area under the curve) values is shown in FIG. 2D.

[0052] Figure 3 shows the impact of an exemplary anti-PD 1-pro-IL- 18 antibody, an exemplary anti-PDl- mutant-IL-15 antibody and an anti-PD 1 antibody incorporating both the aforementioned exemplary pro-IL- 18 and mutantIL- 15, on IFNy release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (PeliCluster CD3, which is capable of crosslinking CD3 and activating T cells independent of FcyR binding) for 72 hours. A summary table of EC50, Emax and AUC (area under the curve) values is shown below the x-y plot.

[0053] Figures 4A-4B show the capacity of an NKG2D x ROR1 bispecific antibody (bsAb) with or without the incorporation of an exemplary? pro-IL18 and IL 15 mutant to induce NK cell mediated tumor cell killing and IFNy release. FIGs 4A and 4B are non-linear x-y plots of tumor cell killing versus test article concentration and IFNy release versus test article concentration, respectively. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.

[0054] Figures 5A-5B show the impact of targeting an exemplary IL-15 variant and an exemplary pro-IL18 toNKG2D on NK cell expansion in PBMC. FIG. 5 A and FIG. 5B are x-y? plots of NK cell percentages and NK cell numbers measured on day 12, respectively, illustrated as a function of a titration of each test article. Relative to tire Fuse926, 100 nM of Fuse916 induced about a 6-fold expansion ofNK cells.

[0055] Figures 6A-6B show the impact of targeting an exemplary IL- 15 variant and an exemplary pro-lL 18 toNKp46 on NK cell expansion in PBMC.

[0056] Figures 7A-7B show7the capacity of an exemplary IL- 15 variant and / or an exemplary pro-IL 18 targeted to y982 T cells via a Vy9V52 TCR x R0R1 bsAb cy tokine fusion to induce expansion of y952 T cells in human PBMC. FIG. 7A and FIG. 7B are x-y plots of y982 T cell percentages and y982 T cell numbers measured on day 12, respectively, illustrated as a function of a titration of each test article. Relative to the Fuse966, 100 nM of Fuse923 induced about a 12- fold expansion ofy982 T cells.

[0057] Figures 8A-8B show the capacity of an exemplary IL- 15 variant and / or an exemplary pro-IL 18 targeted to y982 T cells via a Vy9V82 TCR x R0R1 bsAb cytokine fusion to induce y982 T cell mediated IFNy release and tumor cell killing. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.

[0058] Figures 9A-9C shows the impact of targeting an exemplary IL- 15 variant and an exemplary IL 18 variant to NKp30 plus or minus NKp46 on NK cell expansion from PBMC. Both NK cell frequency and numbers were measured after 21 days of incubating PBMC from a healthy human donor with 100 pM of the test articles described below.

[0059] Figures 10A-10B show? the agonist activity? of y982 TCR specific VHH fused to tire Fc domain of human IgGl to induce redirected lysis of P815 cells stably transduced with eGFP and firefly luciferase (P815) by expanded y982 T cells. Non linear x-y plots of killing percentage as a function of test article concentration is shown. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below? each x-y plot.

[0060] Figures 11A-11B show the capacity of an exemplary? IL- 15 variant and / or an exemplary? pro-IL 18 targeted to y982 T cells via a Vy9V82 TCR x R0R1 bsAb cytokine fusion to induce y982 T cell mediated IFNy release and tumor cell killing. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing(FIG. 11 A) or IFNy release (FIG. 1 IB). Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.

[0061] Figures 12A-12C show tire agonist activity of human NKp46 (FIGs 12A-12B) and human NKp30 (FIG. 12C) specific VEH fused to the Fc domain of human IgGl to induce redirected lysis of P815 cells stably transduced with eGFP and firefly luciferase (P815) by expanded y962 T cells. Non linear x-y plots of killing percentage as a function of test article concentration is show n . Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.

[0062] Figures 13A-13B show' tire capacity of an exemplary IL-15 variant and / or an exemplary pro-IL18 taigeted to human NK cells via a NKp46 x R0R1 bsAb cytokine fusion to induce NK cell mediated IFNy release and tumor cell killing. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 13A) or IFNy release (FIG. 13B). Relative to Fusel 125, the incorporation of cytokine(s) into the bsAb enhanced both tumor cell killing and IFNy release in the rank order of Fusel 124>Fusel 127>Fuseel 126 indicating that the combination of IL-18 and IL-15 was the most potent. The differential readout between test articles was more apparent for IFNy release compared to tumor cell killing. This is likely due to the high E:T ratio of 3: 1 at w'hich maximum tumor cell killing may be reached at a low er NK cell signaling threshold than IFNy release. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.

[0063] Figures 14-15 show the capacity of an exemplary IL-15 variant and an exemplary pro-IL18 targeted to human NK cells via a (1) human NKp30 x human R0R1 bsAb cytokine fusion (Fuse 1147 and Fuse 1145) or (2) NKp30 x NKp46 R0R1 bsAb cytokine fusion (Fusel 148 and Fusel 146), to induce NK cell mediated IFNy release and tumor cell killing. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 14) or IFNy release (FIG. 15). Summary tables of EC50, Emax and AUC (area under the curve) values are shown below' each x- y plot.

[0064] Figure 16 depicts an exemplary fusion protein construct having an IL-15 variant in accordance with various embodiments of the invention.

[0065] Figure 17A-17C depict the functional activity of IL-15 variants as it relates to the induction and release of secreted embryonic alkaline phosphatase (SEAP) by HEK-Blue IL-2 reporter cells.

[0066] Figure 18A-18B depict the functional activity of IL- 15 variants as it relates to T cell mediated IFNy release induced by PD1 targeted IL-15 variants in tire presence of a low "suboptimal" concentration of anti-CD3.

[0067] Figure 19 depicts the functional activity of IL- 15 variants as it relates to T cell mediated IFNy release induced by PD1 targeted IL-15 variants in the presence of allogeneic dendritic cells (DC).

[0068] Figure 20A-20M depict illustrations of formats for pro-IL-18 fusion proteins. All proteins incorporate a human IgG scaffold that is either w ild type homodimeric IgGl or comprising a knob-in-hole (KM) heterodimeric IgGl Fc. Pro-IL-18, when included in the fusion protein is fused as (a) a singular cassette to either the C-tenninal end of the knob (or hole) chain or (b) as singular cassettes to both the C-terminal ends of the knob chain and tire hole chain.Cleavage sites, when present in the pro-IL-18 cassette, are indicated as a star shape. A tumor associated antigen binding domain (TAA), when present, is fused as a singular cassette to the N-terminal end of a knob (or hole) chain, or both chains of tire KIH heterodimer IgG Fc. The TAA binding domain may be a VHH (e.g. EGFR specific clone 9G8) or a fab (e.g. EGFR specific clone C225 from cetuximab).

[0069] Figure 21 A-21 B depict the functional activity as it relates to Antibody Dependent Cellular Cytotoxicity(FIG. 21A) and IFNy release (FIG. 21B) induced by FUSE556 (single armed anti-EGFR VHH 9G8-IgGl), and FUSE686 (single armed anti-EGFR VHH 9G8-IgGl -pro-IL-18).

[0070] Figure 22A-22B depict the functional activity as it relates to Antibody Dependent CellularCytotoxicity (FIG. 22A) and IFNYrelease (FIG. 22B) induced by of FUSE556 (single armed anti-EGFR VHH 9G8- IgGl; targets both EGFR and FcyR), or the combination of FUSE556 and FUSE422 (IgGl-pro-IL-18; only targets FcyR).

[0071] Figure 23 depicts the functional activity as it relates IFNy release induced by of FUSE691 (nivolumab), FUSE645 (EGFR taigeted IgGl-pro-IL-18; non-targeted in this system), FUSE694 (nivolumab-pro-IL-18) and human recombinant IL- 18 on T cell / allogeneic DC co-culture.

[0072] Figure 24A-24B depict the functional activity as it relates to Antibody Dependent Cellular Cytotoxicity ofFUSE556 (single armed anti-EGFR VHH 9G8-IgGl), and FUSE516 (single anned anti-EGFR VHH 9G8-IgGl-pro- IL-18). Graphs illustrating killing of EGFR+ MDA-MB-231 tumor cells by PBMC from each of four normal human donors are shown in FIGs 24A-24B.

[0073] Figure 25A-25B depicts tire functional activity as it relates to pro-IL-18 fusion protein inducedAntibody Dependent Cellular Cytotoxicity (FIG. 25A), IFNy release (FIG. 25A), Granzymc B release (FIG. 25B), and IL-18 release / consumption (FIG. 25B) after 48 hours of co-culture of PBMC and EGFR+ MDA-MB-231 tumor cells. The pro-IL-18 fusion proteins tested were FUSE556 (single armed anti-EGFR VHH 9G8-IgGl), and FUSE516 (single armed anti-EGFR VHH 9G8-IgGl-pro-IL-l 8).

[0074] Figure 26A-26B depicts the functional activity as it relates to Antibody Dependent CellularCytotoxicity induced by FUSE556 (single armed anti-EGFR VHH 9G8-IgGl), variants of pro-IL-18 fused to the C- tenninus of FUSE566 with different protease cleavage sites between the Fc domain and pro-IL-18 (FIG. 26 A), and a variant that contains the PGLALA mutation in its Fc domain to abolish targeting to FcyRs (FUSE627: FIG. 26B). The protease sites tested were Granzymc A (FUSE658), Granzyme B (FUSE516), MMP2 / 9 (FUSE659), and the combmation of Granzyme A, Granzyme B and MMP2 / 9 (FUSE660).

[0075] Figure 27A-27B depicts tire functional activity as it relates to pro-IL-18 fusion protein inducedAntibody Dependent Cellular Cytotoxicity (FIG 27A) and IFNy release (FIG 27B) after 48 hours of co-culture of expanded NK cells and EGFR+ MDA-MB-231 tumor cells at E:T ratios of 1: 1 and 1:5. The pro-IL-18 fusion proteins tested were FUSE556 (single anned anti-EGFR VHH 9G8-IgGl), and FUSE516 (single armed anti-EGFR VHH 9G8- IgGl-pro-IL-18).

[0076] Figure 28A-28B depicts the functional activity as it relates to pro-IL-18 fusion protein inducedAntibody Dependent Cellular Cytotoxicity' (FIG 28A) and IFNy release (FIG 28B) after 48 hours of co-culturc of expanded NK cells and EGFR+ MDA-MB-231 tumor cells at E:T ratios of 2: 1. The pro-IL- 18 fusion protein tested was FUSE555 (dual armed anti-EGFR Fab C225-IgGl-pro-IL-18), which was compared to cetuximab.

[0077] Figure 29A-29B depicts the functional activity as it relates to pro-IL- 18 fusion protein induced Antibody Dependent Cellular Cytotoxicity (FIG 29 A) and IFNy release (29B) after 48 hours of co-culture of expanded NK cells and HER2+ T47D tumor cells at E:T ratios of 5: 1. The pro-IL-18 fusion proteins tested were FUSE624 (dual armed anti-HER2 Fab IgGl -pro-IL- 18) and trastuzumab (Herceptin).

[0078] Figure 30 shows the assessment of the capacity of an anti-PDLl antibody incorporating a pro-IL- 18amut2 variant, designed so that it cannot be cleaved by Granzyme B, to induce T cell mediated IFNy release. A summary table of EC50, Emax and AUC (area under the curve) is shown beneath of x-y plot.

[0079] Figure 31A-31B shows the assessment of the capacity of an anti-PDl antibody, incorporating one of two pro-IL-18 variants (pro-IL 18amut2 or pro-IL18amut9). designed so that it cannot be cleaved by Granzyme B, to induce T cell mediated tumor cell king (FIG. 27 A) and IFNy release (FIG. 27B). A summary table of EC50, Emax and AUC (area under the curve) is shown beneath each x-y plot.

[0080] Figure 32 shows the capacity of several IL- 18 variants to induce non-taigeted trans based release ofSEAP from HEK-Blue-IL-18.

[0081] Figure 33A-33B shows the capacity of several IL-18 variants targeted to human PD-1 to induce cis based release of IFNy from human PBMC derived from a healthy human donor (FIG. 29 A) or human T cells (FIG. 29B).

[0082] Figure 34A-34B shows the capacity of an exemplary IL- 18 variant targeted to mouse PD-1 to mediate tumor growth inhibition of an aggressive syngeneic melanoma tumor in a fully immunocompetent mouse tumor model.

[0083] Figure 34C show the number of different lymphocyte subsets per gram of tumor was assessed from tumor bearing mice treated with PBS (light grey), anti-PD 1 (black), and Fuse 1113 (horizontal lines).

[0084] Figure 34D depicts the frequency of total myeloid cells (CDl lb+ cells), M2 macrophages (CDllb+ F4-80+ CDl lc-CD206+ cells) and the ratios of CD8+ T cells to total Myeloid Derived Suppressor Cells (MDSC; CD1 lb+ Ly6C+ Ly6G- [M-MDSC] and CD1 lb+ Ly6C-mid Ly6G+ [G-MDSC]) . CD8+ T cells to M2 macrophages. anti-PD-1 responsive CD8+ T cells to MDSC, and CD8+ T cells to CD4+ T cells.

[0085] Figure 35A-35B shows the capacity of an exemplaiy IL-18 variant targeted to mouse PD-1 to mediate tumor growth inhibition in a syngeneic colorectal cancer tumor in a fully irrununocompetent mouse tumor model.

[0086] Figure 36A-36B shows he capacity of an exemplary IL- 18 variant taigeted to mouse PD-1 to mediate tumor growth inhibition in a syngeneic rapidly growing colorectal cancer tumor in a fully immunocompetent mouse tumor model.

[0087] Figure 37A shows C57BL / 6 mice injected subcutaneously with MC38i. Serum was collected at 0, 24,72 and 144 hours after the Day 0 treatment and mouse IFNy measured.

[0088] Figure 37B shows C57BL / 6 mice that harbored MC38i tumors and experienced a CR following treatment with aPDl-HT18cis or aPD-l-IL-18 were re-challenged with MC38i after 90 days.

[0089] Figure 38 shows BALB / c mice that harbored CT26.C tumors and experienced a CR following treatment with aPDl-HT18cis were rechallenged with CT26.C after 65 days on the left flank and parental CT26 after 70 days on the right flank. As a means of comparison, a group of 5 naive mice were subjected to the equivalent tumor inoculation.

[0090] Figure 39 shows C57BL / 6 mice were injected subcutaneously with B16-F10R. When mean tumor volume ranged between 75-100 mm3, mice were randomized into groups of 5 and treated with PBS, aPD-1, aPDl- HT18cis on Days 0, 3 and 6 at 15mg / kg. Mice that experienced a CR following treatment with aPDl-HT18cis were rechallenged with B 16-F10 after 60 days.

[0091] Figure 40 shows C57BL / 6 mice harboring B 16-F 10R tumors were treated on Days 0 and 3. Mice were euthanized on Day 5, tumors collected and dispersed into singe cell suspensions for flow cytometnc assessment of TIL (CD45+ cells). TEM and TCM were defined CD44+ / CD62L- and CD44+ / CD62L-, respectively within flic CD8+ pool. aPD-1 responsive CD8+ T cells were defined as PD1+ / TCF1+ cells within the CD8+ pool.

[0092] Figure 41 shows C57BL / 6 mice harboring B 16-F 1 OR tumors were treated on Days 0 and 3. Mice were euthanized on Day 5, tumors collected and dispersed into singe cell suspensions for flow cytometric assessment of TIL (CD45+ cells). All myeloid subsets were gated on CDl lb. Within that pool, MDSC were defined as Ly6G+ / Ly6Cmid+Ly6G- / Ly6Cliigh and M2 macrophages as F480+ / CD206+.

[0093] Figure 42A and 42B depicts the impact of exemplary anti-PDl-pro-IL-18 antibodies containing different IL-18 variants on IFNv release mediated by PBMC activated with PeliCluster CD3 for 48 hours, washed and the re-exposed to a 10 fold lower concentration of PeliCluster CD3 for an additional 48 hours.

[0094] Figure 43 (panels A-E) depicts exemplary’ fusion proteins in which the N-terminus of pro-IL- 18 is fused to the C-terminus of the knob of a knob-into-hole heterodimeric IgGf protein; which has a structure from N- to C- tenninus comprising knobs-in-hole (KiH) Fc - propeptide (PP) - enterokinase-cleavable site (EK) - IL- 18 wild type or its variants. This depicts exemplary fusion proteins such as IDs: FUSE-480, FUSE-481, and FUSE-442 in Table 6. Further modification was made to pro-IL- 18 to reduce aggregation of the molecule, wherein each cysteine residue in both the propeptide and mature IL-18 was replaced with serine (as in FUSE-480, denoted as “IL-18AS”), with alanine (as in FUSE- 481, denoted as “IL-18AA”), or with valine (as in FUSE-442, denoted as “IL-18AV”). Alternatively, fire N-tenninus of pro-IL- 18 can be fused to the C-tenninus of the hole chain of a KiH heterodimeric IgGl proteins. The biological activity defined as the EC50-SEAP for each compound is shown in panel E.

[0095] Figure 44 (panels A-E) depicts exemplary fusion proteins in which the N-terminal of pro IL-18 was fused to the C-terminal of an IgGf CH3 domain (which is also a knob chain of a knob-into-hole heterodimeric IgGl protein as in Figure 43), and the pro IL- 18 incorporated four amino acid substitutions hypothesized to reduce binding to IL-18BP while maintaining wild type binding to the IL-18 receptor complex, denoted as “pro-IL-18mut2'’. These fusionproteins have a stmcture from N- to C-terminus comprising knobs-in-hole (KiH) Fc - propeptide (PP) - enterokinase- cleavable site (EK) - IL-18mut2. Further modification was made to the pro-IL-18mut2 to reduce aggregation of the molecule, wherein each cysteine residue in both the pro-peptide and mature IL-18mut2 was substituted with serine (denoted as ‘TL-18mut2AS'’, as in FUSE-422; panel B), with alanine (denoted as “IL-18mut2AA”, as in FUSE-423; panel C). or with valine (denoted as “IL-18mut2AV”. as in FUSE-424; panel D). The biological activity defined as the EC50-SEAP for each compound is shown in panel E.

[0096] Figure 45 (panels A-D) depicts exemplary fusion proteins with (panel A) or without (panel B) the propeptide to examine the impact on masking of “IL-18AV” biological activity (panel C), wherein Fc fusion variants were generated incorporating “'IL- 18 AV" with the propeptide (panel A; FUSE-442) or without (panel B; FUSE-505) the propeptide. The biological activity defined as the EC50-SEAP for each compound is shown in panel D.

[0097] Figure 46 (panels A-D) depicts exemplary fusion proteins with (panel A) or without (panel B) the propeptide to examine the impact of propeptide on masking of ’iL-18mut2AV" biological activity, wherein Fc fusion variants were generated incorporating “IL-18mut2AV” without the propeptide (lienee, a mature IL-18 with mutation, denoted as “matIL-18mut2-AV”, see panel B; FUSE-441) or with the propeptide (panel A; FUSE-424). For a fusion protein devoid of the propeptide, the EK cleavage site tiiat replaced the Caspase 1 site was moved to a position direcdy in between the CHS domain of the knob and the mature IL- 18 AV without the addition of a flexible linker. Panel C depicts the activation readout using the HEK-Blue IL-18AV reporter cell assay following exposure to a titration of FUSE-441 (Fc-EK-IL- 18 AV) or FU SE-424 (Fc-EKpp-IL- 18 AV) with or without treatment with EK. The biological activity defined as the EC50-SEAP for each compound is shown in panel D.

[0098] Figure 47 (panels A-D) depicts exemplary fusion proteins in which the N-tenninus of pro-IL-18 is fused to the C-terminus of IgGl Fc protein or IgG4 Fc; which has a structure from N- to C-tenninus comprising IgGl Fc- propeptide (PP) - IL-18AV (FUSE-507; panel A) and IgG4 Fc- propeptide (PP) - IL-18AV (FUSE-509; panel B). Panel C depicts the activation readout using the HEK-Blue IL- 18 AV reporter cell assay of exposing the cells to a titration of FUSE-507 or FUSE-509 with or without treatment with EK. The biological activity defined as the EC50-SEAP for each compound is shown in panel D.

[0099] Figure 48 (panels A-E) depicts exemplary fusion proteins in which the N-tenninus of pro-IL- 18 is fused to the C-terminus of HSA with or without the propeptide (PP); which has a structure from N- to C-terminus comprising HSA- propeptide (PP) - IL-18AV (FUSE-501; panel A) and HSA- IL-18AV (FUSE-503; panel B). Panels C and D depict the activation readout. The biological activity defined as the EC50-SEAP for each compound is shown in panel E.

[0100] Figure 49 (panels A-E) depicts exemplary fusion proteins in which the N-tcmiinus of pro-IL- 18mut2 is fused to the C-terminus of HSA with or without the propeptide (PP); which has a structure from N- to C-tenninus comprising HSA- propeptide (PP) - IL-18mut2AV (FUSE-502; panel A) and HSA- IL-18mut2AV (FUSE-504; panel B). Panels C and D depict the activation readout. The biological activity defined as the EC50-SEAP for each compound is shown in panel E.

[0101] Figure 50 (panels A-D) depicts exemplary fusion proteins in which the C-terminus of pro-IL-18 is fused to the N-terminus of the knob of a knob-into-hole heterodimeric IgGl protein with or without the propeptide (PP); which has a structure from N- to C-tcrminus comprising propeptide (PP) - IL-18AV - knobs-in-holc (KiH) Fc (FUSE- 499; panel A) and IL-18AV - knobs-in-hole (KiH) Fc (FUSE-500; panel B). Panel C depicts the activation readout. The biological activity defined as the EC50-SEAP with or without exposure to Caspase 1 for each compound is shown in panel D.

[0102] Figure 51A and 5 ID depicts exemplary fusion proteins with a structure from N- to C- terminus: Fc- ppMMP2 / 9-cleavage sites-IL-18-AV (FUSE-486), Fc-ppMMP9 / 2-cleavage sites-IL-18-AV (FUSE-487), in which the cleavage sites are specific for the metalloproteases, MMP2 and MMP9, with preferred enzyme to the left of the forwardslash, or FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV). Figure 5 IB depicts the activation readout relating to FUSE-486 and FUSE-487, with or without MMP2 treatment. The biological activity defined as the EC50-SEAP for each of FUSE-486 and FUSE-487, with or without MMP2 treatment, is shown in figure 5 IB. FIG. 51C shows that FUSE587 w as about 3,000-fold attenuated relative to recombinant human IL-18. Interestingly, we observed that cleavage of FUSE587 with MMP2 released and IL-18AV variant that was still about 100-fold attenuated relative to recombinant IL- 18. In contrast, cleavage with Granzyme B released an IL- 18 AV variant with activity similar activity as recombinant IL-18. Cleavage with Granzyme B results in release of mature IL- 18 AV without any N-terminal residues constituting an overhang, whereas 11 and 15 amino acid N-terminal polypeptide overhangs remain after cleavage of FUSE486 and FUSE587, respectively, with MMP2. We speculated that these overhangs might be attenuating IL-18AV activity; albeit to a lesser degree than the full size variant propeptide. This phenomenon was further investigated in FIG. 53 and FIG. 55. Figures 5 ID and 51G also depicts exemplary fusion proteins with a structure from N- to C- terminus: Fc-ppGb-cleavage sites-IL-18-AV (FUSE-485; B-19D) or Fc-ppGb-cleavage sites-IL-18mut2- AV (FUSE-462; 51D) or Fab-Cetuximab-Fc-ppGb-cleavage sites-IL-18-AV (FUSE-517; 51G). in which the cleavage site is specific for granzyme B (Gb). Figure 5 IE depicts the activation readout relating to FUSE-462 and FUSE-485, with or without granzy me B treatment. Figure 5 IF shows the biological activity defined as the EC50-SEAP for each of FUSE- 462 and FUSE-485, with or without granzyme B treatment. Figure 51H depicts the activation readout relating to FUSE- 517, with or without enzyme treatment. Figure 511 shows the biological activity defined as the EC50-SEAP for FUSE- 517, with or without granzyme B treatment.

[0103] Figure 52 (panels A-G) depicts the impact of IL-18BP on the biological activity of recombinant human IL-18 (rhIL-18) and the EK cleavage products of the exemplary’ fusion proteins, Fc-ppEK-IL-18-AV (FUSE-442) and Fc-ppEK-IL-18mut2AV (FUSE-424). The biological activity defined as the EC50-SEAP for each compound with and without the addition of IL-18BP (competition assay) is shown in panels B, D and F, with corresponding biological activity defined as the EC50-SEAP shown in panels C, E, and G, respectively.

[0104] Figure 53 (panel A) depicts diagrams of exemplary fusion proteins in which the C-tenninus of pro-IL- 18 is fused to the N-terminus of the knob of a knob-into-hole heterodimeric IgGl protein with different size polypeptidesfused to the N-terminus of mature IL-18. Figure 53 (panel B) depicts the biological activity of each fusion protein using the IL-18 reporter cell line, HEK-Blue IL- 18.

[0105] Figure 54A, 54B(i), 54B(ii), 54C, 54D(i), 54D(ii) and 54E depict human IL-18 engineered mutant fusion proteins in accordance with various embodiments of the invention.

[0106] Figure 55 shows the impact of the size of polypeptides fused to the N-terminus of mature IL- 18 on the biological activity of a single IL-18AV fused to the N-terminus of IgGl Fc.

[0107] Figure 56A-56H shows the impact of the substituting the cysteine residue in the pro-peptide and cysteine residues in tire mature IL 18, which were fused together to fonn the pro-IL-18 variant cassette, on tire biological activity of each variant using the HEK Blue IL18 assay system.

[0108] Figure 57A-57B shows the impact of targeting pro-IL18 to within close proximity of its receptor complex (i.e., “cis activity).

[0109] Figure 58A-58E depict schematics of multiple fonnats of anti-RORl / CD3 bispecific antibodies, also tenned T cell engagers (TCEs), illustrating protein designs of TCE fusion proteins comprising a knob-in-hole (KIH) heterodimeric IgGl Fc scaffold with (1) a CD3-specific fragment antigen-binding region (Fab) on the N-terminal end of a knob chain (shown in panels 58A. 58B, 58D and 58E) or a hole chain (shown in 58C) of the KIH. and (2) one or more VHH or single-chain variable fragment (scFV) specific for a tumor-associated antigen (TAA) (eg., ROR1) in one or more positions. The examples shown illustrate the TAA binder linked to (a) the N-terminal end of the hole chain in tandem (58 A), (b) the N-terminal end of tire hole chain and on the C-terminal end of the CD3 -specific (anti-CD3) Fab light chain (58B), (c) the N-terminal end of the knob chain and on the C-terminal end of the CD3 -specific (anti-CD3) Fab light chain (58C). (d) the N and C-terminal end of the hole chain (58D) and (e) tire N-terminal end of the hole chain only (58E).

[0110] Figure 59A-59B show the production yield (59A) and the melting temperature (Tm) (2B) of exemplary' fusion proteins (denoted as a FUSE ID, “FUSE-XXX”).

[0111] Figure 60A-60D shows the apparent binding affinity of exemplary TCEs for cell membrane expressedCD3 in human T cells and in non-human primate (NHP), Cynomolgus T cells.

[0112] Figure 61 A-61 E depict the apparent binding affinity of exemplary' TCEs for cell membrane expressedR0R1 using a series of engineered MDA-MB-231 variant cell lines expressing different cell surface densities of the R0R1. Figure 61F-6 IK depict the capacity of exemplary TCEs specific for R0R1 to bind the membrane distal R0R1 Ig domain.

[0113] Figure 62A-62D depict the binding affinity (KD) for RORL determined via Bio-layer Interferometry(BLI), of TCEs: FUSE-211 (in FIG. 62A), FUSE-393 (in FIG. 62B), and FUSE-394 (in FIG. 62C), with therr calculated binding affinity shown in FIG. 62D.

[0114] Figure 62E-62G depict tire binding affinity for ROR1 of a 2A11 VHH - Fc fusion protein (denoted asFUSE-112. in FIG. 62E) and of a 2A11 VHH mutant - Fc fusion protein (denoted as FUSE-453, in FIG. 62F), with tiieircalculated binding affinity shown in FIG. 62G, which demonstrated the impact of replacing the cysteine residues in CDR1 and CDR3 of the RORl-specific VHH.

[0115] Figure 62H-62J depict the binding affinity for R0R1 of a 5A1 VHH - Fc fusion protein (denoted asFUSE-179, in FIG. 62H) and of a 5A1 VHH mutant - Fc fusion protein (denoted as FUSE-454, in FIG. 64), with their calculated binding affinity shown in FIG. 62J. which demonstrated the impact of replacing the cysteine residues in CDR1 and CDR3 of the RORl-specific VHH.

[0116] Figure 63A-63D illustrate TCEs induction of human peripheral blood mononuclear cells (PBMC)- mediated, R0R1 -dependent cytotoxicity (characterized as percentage killed) of MDA-MB-231 tumor cells, wherein the tested TCEs include FUSE-211, FUSE-393, and FUSE-394 and the human PBMC were from donor 1 (FIG. 63A), donor 2 (FIG. 63B), or donor 3 (FIG. 63C). FIG. 63D summarizes the calculated EC50 of each TCE for inducing a killing percentage of 50% of the tumor cells.

[0117] Figure 63E-6H illustrate TCEs induction of IFN-y release from human PBMC (characterized as concentration of IFN-y in cell culture supernatant) in the presence of MDA-MB-231 tumor cells. The tested TCEs include FUSE-211, FUSE-393, and FUSE-394, and the human PBMC were from donor 1 (FIG. 63E), donor 2 (FIG. 63F), or donor 3 (FIG. 63G). FIG. 63H summarizes the calculated EC50 of each TCE for inducing 50% of a maximum IFN-y release.

[0118] Figure 631-630 illustrate the capacity of FUSE-211, FUSE-393, and FUSE-394 to mediate reducedR0R1 -dependent IFN-y release from human PBMC (average of three donors) or Pan-T cells while maintaining cytotoxic efficacy (maximum killing of MDA-MB-231), or “decoupling of cytotoxicity from cytokine release’’ compared to a FUSE-277, a prototypical highly potent TCE tiiat serves as the “non-decoupling" control TCE.

[0119] Figure 63P-63V depict ROR1 dependent killing, induction of cytokine release and calculated decoupling ratios following 24, 48 and 72 hour co-cultures of PBMC effectors with ROR1+ MDA-MB-231 tumor taigets.

[0120] Figure 64A-64C illustrates TCEs induction of human PBMC-mediated, R0R1 -independent cytotoxicity of T47-D tumor cells (which do not express R0R1), characterized as IFN-y concentration in the co-culture supernatant. Panels A-C show the results wherein the human PBMC were from donor 1 (panel A), donor 2 (panel B). and donor 3 (panel C), respectively.

[0121] Figure 65A-65E and 65G-65K depict TCEs induction of human PBMC-mediated, R0R1 -dependent cytotoxicity and cytokine release, respectively, as a function of tire cell surface density of R0R1. Data was generated using a series of cell lines of different origins expressing different numbers of R0R1 molecules on their cell surfaces. The cell lines are shown in a descending order of cell surface R0R1 density, from high to low. That is. NCCIT (FIG. 65 A, G), MDA-MB-231 (FIG. 65B,H), NCI-H1975 (FIG. 65C,I), DU-145 (FIG. 65D,J) and R0R1 -negative T-47D (FIG. 65E,K). FIG. 65F and 65L summarize the potencies for cytotoxicity and cytokine release induced by each of the TCEstowards a series of cell lines with different cell-surface R0R1 densities. FIG. 65M illustrates the calculated decoupling ratio for each TCE across the cell line series.

[0122] Figure 66A-66E and 66G-66K depict TCEs induction of human PBMC-mcdiatcd, R0R1 -dependent cytotoxicity and cytokine release, respectively, as a function of the cell surface density of R0R1 using a series of engineered MDA-MB-231 variant cell lines expressing different cell surface densities of the R0R1. The surface densities of ROR1 on the engineered MDA-MB-231 were as follows: -2,000,000 molecules per cell (MDA++; FIG. 66A, 66G), -500,000 molecules per cell (MDA+; FIG. 66B, 66H), -35,000 molecules per cell (MDA: FIG. 66C, 661), - 17,000 molecules per cell (MDA-low; FIG. 66D, 66 J), and 0 molecule per cell (MDA-KO; FIG. 66E, 66K). FIG. 66F and 66L summarize tire potencies for cytotoxicity and cytokine release induced by each of the TCEs towards MDA-MB-231 cells with different cell-surface ROR1 densities. FIG. 66M represents the calculated decoupling ratios (cytotoxicity from cytokine release) for each TCE and FIG. 66N is a regression analysis between tire magnitude of IFNy versus RORI cell surface density of a non-decoupling TCE control versus an exemplary decoupling TCE.

[0123] FIGs. 67A and 67B depict TCE-induced expression of activation markers, CD69 and CD25, respectively, on T cells following co-culture of PBMCs with RORI -expressing tumor cells. Fig. 67C summarizes the potency by which each TCE induces CD69 or CD25, defined as the EC50-binding. FIG 67D and FIG. 67F are bar charts illustrating the decoupling of cytotoxicity from tire induction of the cell surface biomarkers of T cell activation, CD69 and CD25. FIG. 67E and FIG. 67G are bar charts illustrating the decoupling of the induction CD69 and CD25 from cytokine release.

[0124] Figure 68A-68C depict the pharmacokinetics (PK) of exemplary TCEs in a single dose of either 5 mg / kg (FIG. 68A) or 0.5 mg / kg (FIG. 68B) administered to wild type C57BL / 6 mice over a 10-day period. FIG. 68C depicts the PK parameters for each test article.

[0125] Figure 69A-169E depict a schematics of two humanized mouse xenograft tumor models (FIGs 69A and 69C). In both cases, the highly aggressive human ROR1+teratoma, NCCIT was injected into immune incompetent mice. Tumor growth over time for the first model is shown in FIG. 69B and second model in FIGs 69D and 69E. In both cases, the test articles consisted of FUSE-394 and the non-decoupled control TCE, FUSE-399.

[0126] Figure 70A-70E depict tire capacity of an exemplary TCE to induce T cell mediated killing of ROR1- positive tumor cells (MDA-MB-231 ; FIG. 70A), ROR 1 negative tumor cells (T-47D; FIG. 70B) and a mixture of ROR 1 - positive tumor cells and RORI negative tumor cells (FIG. 70C and FIG. 70D). For the latter, killing of the RORI positive tumor is shown in FIG. 70C. Killing of the RORI negative tumor is tenned “bystander killing”, the comparison of which to a non-decoupling TCE is shown in FIG. 70D. FIG. 70E depicts IFNy production by the same T cells used to assess “bystander killing” as a function of exemplary TCE concentration.

[0127] Figure 71A-71B depict the (a) percent killing of RORI -positive tumor cells (MDA-MB-231) as a function of T cell numbers (FIG. 71 A) in the context of exemplary TCEs and (b) TCE mediated “serial killing” calculated as the average number of TCE induced RORl-positive tumor cells killed per T cell in a 24-hour period (FIG. 71B).

[0128] Figure 72 depicts the apparent binding affinity (EC50-binding) of the R0R1 specific VHH-Fc incorporating either the non humanized VHH clone 5A1 (FUSE179; black circle), the humanized clone 5A1-FA9-HK1 (FUSE559; black square) or the humanized clone 5A1-HK1 (FUSE524; black triangle) was calculated.

[0129] Figure 73A-73B depict tire affinity (KD) of the ROR1 specific VHH-Fc incorporating either the non humanized VHH clone 5A1 (FUSE179; FIG. 73A) or the humanized clone 5A1-FA9-HK1 (FUSE559; FIG. 73B) was calculated. FIGs. C-74A-74B depict the apparent binding affinity (EC50-binding) of the two bispecific antibodies (bsAbs).

[0130] Figure 75A-75B depict capacity of several ROR1 x CD3 specific bsAbs to induce (1 ; FIG. 75A) T cell mediated killing of ROR1+ MDA-MB-231 tumor cells and (2: FIG. 75B) T cell mediated release of IFNy when comixed with ROR1+ MDA-MB-231 tumor cells.

[0131] Figure 76A-76I depict capacity of several ROR1 x CD3 specific bsAbs to induce (1; FIGs 76A-76D)PBMC mediated killing of four different tumor cell lines and (2; FIGs 76E-76H), PBMC mediated release of IFNy when co-mixed with the aforementioned tumor cells.

[0132] Figure 77A-77F depict capacity of several ROR1 x CD3 specific bsAbs to induce (1: FIG. 77A)PBMC mediated killing of ROR1+ MDA-MB-231 tumor cells and (2; FIG. 77B), PBMC mediated release of IFNy when co-mixed with ROR1+ MDA-MB-231 tumor cells, (3. FIG. 77C) upregulation of cell surface CD69 expression on T cells within PBMC when co-mixed with ROR1+ MDA-MB-231 tumor cells, (4. FIG. 77D) upregulation of cell surface PD-1 expression on T cells within PBMC when co-mixed with ROR1+ MDA-MB-231 tumor cells, and (5. FIG. 77E) upregulation of cell surface TIGIT expression on T cells within PBMC when co-mixed with ROR1+ MDA-MB- 231 tumor cells.

[0133] Figure 78A-78C depict capacity’ of FUSE399 (black reverse triangle) and FUSE608 (black circle) to induce CD8+ T cell mediated killing of a constant number (5000 cells) of ROR1+ MDA-MB-231 cells across a range of CD8+ T cell numbers from 313 to 40,000.

[0134] Figure 79A-22D depict whether FUSE608 induced bystander killing of ROR1 negative tumors comixed w ith ROR1 positive tumors.

[0135] Figure 80A-80B depict the capacity of FUSE608 and tire non-decoupled benchmark ROR1 x CD3 bsAb, FUSE399, to mediate tumor growth inhibition (TGI) in a mouse tumor xenograft model.DESCRIPTION OF THE INVENTION

[0136] All references cited herein are incorporated by reference in their entirety as though fully set forth.Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in tire art to which this invention belongs. Singleton el al., Dictionary of Microbiology and Molecular Biology 3rded, Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 4thed.. Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2'ded. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U. S. Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4.946.778; Bird. Science 242:423-42 (1988); Huston etal., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al. Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9): l 126-36).

[0137] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no waylimited to tire methods and materials described. For purposes of the present invention, the following terms are defined below.

[0138] As used herein the term “about” or “approximately” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%. 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0139] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways tiiat are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be detennined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0140] As used herein the term “immunoglobulin heavy chain constant region” is used interchangeably with the tenn “Fc region” and is understood to mean the carboxyl-terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding an Fc receptor. Each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CHl-hinge-CH2-CH3(- CH4). CH4 is present in IgM, which has no hinge region. The immunoglobulin heavy chain constant region suitable for the invention preferably comprises an immunoglobulin hinge region, and preferably also includes a CH3 domain. The immunoglobulin heavy chain constant region most preferably comprises an immunoglobulin hinge region, a CH2 domain and a CH3 domain.

[0141] As used herein, the term immunoglobulin “hinge region” is understood to mean an entire immunoglobulin hinge region or at least a portion of the immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.

[0142] As used herein, the tenn “vector” is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated into the host cell genome, or to replicate autonomously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an adeno-associated virus.

[0143] As used herein, the term “gene expression” or “expression” of a protein or polypeptide, is understood to mean the transcription of a DNA sequence, translation of the mRNA transcript, and secretion of a protein or polypeptide product. In some embodiments, the expression process also includes or is followed by purification; for example, protein A affinity chromatography or other means such as size exclusion chromatography can be used for purification.

[0144] The temi “linker” with respect to amino acid linker in a polypeptide can be a short peptide, such as a dimer of two amino acids, a tri-mer of three amino acids, a 4-mer of four amino acids, a 5-mer of five amino acids or a peptide selected from the group consisting of T, PT, MPT, S. GS. GGS. GGGS (SEQ ID NO:235), and (GGGGX, (SEQ ID NO:236))n wherein X is Q, A, E or S and n=l-5 or an integer laiger than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))n where n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in lengtii. Additional examples include (X,GGGG (SEQ ID NO:317))n wherein X, is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5, (GX,GGG (SEQ ID NO:31 ))nwherein X,. is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5, (GGX GG (SEQ ID NO:319))nwherein X-,. is Q, A. E or S and n=l-5 or in some embodiments, an integer laiger than 5, (GGGX G (SEQ ID NO:320))n wherein X-, is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. Still additional examples include (X,GGG)n wherein X’,. is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5, (GX,GG)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGX, G)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5, (GGGXQn wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5. Still additional examples include (X,GG)n wherein Xkis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GX,G)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGXjn wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments. Still additional examples include (X,G)n wherein Xλ is Q, A, E or S and n=I -5 or in some embodiments, an integer laiger than 5, (GXQn wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. As further examples, the short polypeptide is 1, 2, 3, or 4 amino acids, or flexible linkers or amino acid chain of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20. 25, 30, or 35 amino acids. In some embodiments, the amino acid linker is an IL-18 propeptide or IL- 18 propeptide variant. In some embodiments, the amino acid linker is a fragment of an IL- 18 propeptide or IL- 18propeptide variant; for example, about 30-36 amino acids in length, about 5-10, 11-20, 21-30, or 31-40 amino acids in length.

[0145] As used herein, the term “cis” in the context of interactions refers to tire interaction between two molecules on the same cell, such as proteins expressed on the same cell. A nonlimiting example is the interaction between B7-1 and PD-L1. “Cis” interaction as used herein also includes multispecific proteins that bind two proteins on the same cell simultaneously.

[0146] As used herein, the term “trans” in the context of interactions refers to the interaction betw een two molecules on different cells, such as proteins on different cells. A nonlimiting example is the interaction between the prototypical interaction of the T cell Receptor (TCR) and its ligand, an MHC-I / peptide complex. ‘Trans” interaction as used herein also include multispecific proteins that bind one protem on one cell and a second protein on a separate cell; hence a “bridge” would be considered as occurring in trans. Also included as a “trans” interaction is an interaction between a soluble protein and a cell surface protein, although not forming a cellular bridge.

[0147] As used herein, TL-18 fusion protein” refers to a fusion protein that includes wild-type IL- 18 or IL- 18 variants, unless specifically noted as only including the w ild-type IL- 18, or only including the IL- 18 variant. Thus, in particular embodiments, the TL-18 fusion protein” oidy includes any one of the IL- 18 variants as described herein.

[0148] “Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the tenn “antibody” are VHH antibodies or nanobody antibodies that consist of antigen binding fiagment(s) of heavy chain only antibodies fused to an Fc, where the VHH are preferably derived from camelids, polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies.

[0149] “Antigen-binding fragments” or “bispecific antigen-binding fragments” are any proteinaceous structure that may exhibit binding affinity' for a particular antigen. Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments arc composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen-binding fragments include, without limitation, diabodies. Fab, F(ab’)2, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody, bivalent fragments comprising tw o Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CHI domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, adAb fragment (e.g., Ward et al.. Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (e.g., Holt et al; Trends Biotechnol. 2003November; 21(11):484-90); camelid VHH or nanobodies (e.g., Revets et al; Expert Opin Biol Ther. 2005 January; 5(1): 111-24); an isolated complementarity determining region (CDR), and tire like. All antibody isot pcs may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds (e g., a receptor-ligand pair; for example, a PDl-Fc fusion would target PDL1 and PDL2). Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase ’‘an antibody or antigen-binding fragment thereof’ may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0150] “VHH” refers to the single variable domain on a heavy chain. It may also be termed as a nanobody.The VHH (or nanobody) includes three CDR domains that make up the majority of tire paratope or antigen binding fragment of heavy chain only antibodies (HcAb). Generally, HcAb is naturally produced by camelids and sharks.

[0151] “Single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short flexible linker peptide (generally about ten to 25 amino acids long).

[0152] hr pharmacokinetics study, half-life (t 1 / 2) generally refers to the time that the drug concentration needs to decrease by 50%. “Tl / 2 alpha” refers to the half-life of distribution phase, w hereas “Tl / 2 beta” refers to the half-life of elimination phases. By some descriptions, the alpha half-life is the rate of decline in plasma concentrations due to the process of drug redistribution from the central to the peripheral compartment, and the beta half-life is the rate of decline due to the process of dmg elimination due to metabolism. During the distribution phase, changes in the concentration of drug in plasma reflect primarily movement of dmg from the circulation to internal compartments, rather than loss from, tire body. However, once tire dmg in the plasma and tissues has reached equilibrium, the decline of plasma concentration is driven by elimination of the dmg from the body, called the elimination phase (late phase).

[0153] “ROR1” (Receptor Tyrosine Kinase-Like Orphan Receptor 1) refers to the 106-kDa member of the receptor tyrosine kinase family having a UniProt Accession Number Q01973 (human) and Q9Z 139 (mouse) .

[0154] The tenn “CD3” refers to the human CD3 protein multi-subunit complex. The CD3 protein multisubunit complex is composed to 6 distinctive polypeptide chains. These include a CD3y chain (e.g., SwissProt P09693). a CD35 chain (e.g., SwissProt P04234), two CD3a chains (SwissProt P07766), and one CD3 chain homodimer (e.g., SwissProt 20963), and which is associated with the T cell receptor a and 0 chain. CD3 is further clustered with the T cell receptor (TCR) or TCR / CD3 complex, which represents the key activation receptor expressed on T cells. Thus, crosslinking of CD3 or the TCR result in similar signaling pathways and activation of T cells. The term “CD3” includes any CD3 variant, isoform and species homolog which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides, unless noted.

[0155] “Immunospecifically” when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopesof a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Phrases such as '‘anti-[antigen] antibody” (e.g., anti-RORl antibody) or “[antigen] -specific antibody” (e.g., RORl-specific antibody” are meant to convey that the recited antibody specifically binds the recited antigen.

[0156] “Isolated” means a biological component (such as an antibody) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Antibodies that have been “isolated” thus include antibodies purified by standard purification methods. “Isolated antibodies” can be part of a composition and still be isolated if such composition is not part of the native environment of the antibody. The term also embraces antibodies prepared by recombinant expression in a host cell as w ell as chemically synthesized antibodies.Fusion proteins

[0157] Various embodiments provide for a fusion protein comprising a scaffold polypeptide and at least two of: (a) one or more of an interleukin 15 (IL- 15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, and (c) one or more of a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH. In various embodiments, (b) is one or more IL- 18 variants.

[0158] Various embodiments provide for a fusion protein comprising a scaffold polypeptide and at least two of: (a) one or more of an interleukin 15 (IL-15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, and (c) one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA). In various embodiments, (b) is one or more IL-18 variants. Polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a TAA include those described herein, and those known in the art.

[0159] Various embodiments provide for a fusion protein comprising a scaffold polypeptide and at least two of: (a) one or more of an interleukin 15 (IL- 15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL-18 variant, or a fragment of the IL-18 variant, and (c) one or more immune checkpoint-targeting fragment. In various embodiments, (b) is one ormore IL-18 variants.

[0160] In various embodiments, the one or more polypeptides capable of binding the TAA comprise a receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH. In various embodiments, the TAA comprises EGFR, HER2, or DLL. Polypeptides capable of binding the TAA include those described herein and those known in tire art.

[0161] In various embodiments, tire activation receptor comprises cluster of differentiation (CD) 3, CD16, v9TCR, 52 TCR or 51 TCR. or tire costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40. Accordingly, Polypeptides capable of binding these activation receptors or costimulatory receptors include those described herein and those known in the art.

[0162] In various embodiments, the immune checkpoints include but are not limited to PD-1 , PD-L1 , CTLA- 4, LAG-3. In various embodiments, one or more immune checkpoint-targeting fragment comprises immune checkpoint- taigcting fragments of known antibodies. Examples of anti-PDl fragments include fragments (E.g. Fab, Fv) from pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), SymO2L dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, or PF-06801591. Additional examples of anti-PDl fragments also include fragments (e g., Fab, Fv) from vopratelimab, camrelizumab, sintilimab, AMP-224, AMP-514, and Acrixolimab. Examples of anti-PD-Ll antibodies include but arc not limited to garivulimab (BGB-A333), cosibclimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824. Thus, for example, the fusion protein comprises an anti-PDl fragment from an anti- PDl antibody (e.g.. Fab, Fv), an IL-18 variant, and an IL-15 variant and optionally, a linker.

[0163] In various embodiments, the fusion protein comprises the scaffold polypeptide and (a) one or more of an interleukin 15 (IL- 15) variant, and (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, In various embodiments, (b) is one or more IL- 18 variants.

[0164] In various embodiments, the fusion protein comprises the scaffold polypeptide and all tiiree of (a) one or more of an interleukin 15 (IL-15) variant, (b) one or more of an interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, or a fragment of the IL- 18 variant, and (c) one or more of a receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH. In various embodiments, (b) is one or more IL- 18 variants.

[0165] In various embodiments, the fusion protein comprises the scaffold polypeptide and all tiiree of (a) one or more of an interleukin 15 (IL-15) variant, (b) one or more of an interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, or a fragment of the IL- 18 variant, and (c) one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA). In various embodiments, (b) is one or more IL- 18 variants.

[0166] In various embodiments, the fusion protein comprises the scaffold polypeptide and all tiiree of (a) one or more of an interleukin 15 (IL-15) variant, (b) one or more of an interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, or a fragment of the IL- 18 variant, and (c) one or more immune checkpoint-targeting fragment. In various embodiments, (b) is one or more IL- 18 variants.

[0167] In various embodiments, the one ormore oftlie IL-15 variant is 2, 3, 4, 5, or 6, or up to 8 IL-15 variants, In various embodiments, the one or more of the one or more of the interleukin 18 (IL- 18), the fragment of the IL- 18. the IL-18 variant, or the fragment of the IL-18 variant is 2, 3, 4, 5, or 6, or up to 8. In various embodiments, the one or more of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH is 2. 3, 4, 5, or 6. or up to 8.

[0168] In various embodiments, the fusion protein further comprises a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally theactivation receptor comprises cluster of differentiation (CD) 3, CD 16, NKp46, or NKG2D, and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp30, CD2, ICOS, 0X40, CD40L and CD40.

[0169] In various embodiments, tire fusion protein further comprises a linker between tire scaffold polypeptide and the (a) one or more of an interleukin 15 (IL- 15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, or (c) one or more of a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH.

[0170] In various embodiments, the fusion protein further comprises a linker between the scaffold polypeptide and tire (a) one or more of an interleukin 15 (IL- 15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, and (c) one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory' receptor expressed on an immune cell, or one or more polypeptides capable of binding atumor associated antigen (TAA). In various embodiments, (b) is one or more IL-18 variants.

[0171] In various embodiments, the fusion protein further comprises a linker between the scaffold polypeptide and the (a) one or more of an interleukin 15 (IL- 15) variant, (b) one or more of an interleukin 18 (IL- 18), a fragment of tire IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, and (c) one or more immune checkpoint-targeting fragment. In various embodiments, (b) is one or more IL- 18 variants.

[0172] In various embodiments, the IL- 15 variant, the interleukin 18 (IL-18), the fragment of the IL-18, the IL- 18 variant, orthe fragment of the IL-18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, are each independentiy fused to the C-terminus of the scaffold polypeptide.

[0173] In various embodiments, the IL-15 variant, the interleukin 18 (IL-18), the fragment of the IL-18, the IL- 18 variant, orthe fragment of the IL- 18 variant, or tire receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH. are each independently fused to the N-terminus of the scaffold polypeptide.

[0174] In various embodiments, IL- 15 variant, the IL- 18, the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, and the receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or the one or more polypeptides capable of binding a tumor associated antigen (TAA) are each independentiy fused to the C-terminus of the scaffold polypeptide. In various embodiments, (b) is one or more IL- 18 variants.

[0175] In various embodiments, IL- 15 variant, the IL- 18, the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, and the receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or the one or more polypeptides capable of binding a tumor associated antigen (TAA) are each independentiy fused to the N-terminus of the scaffold polypeptide. In various embodiments, (b) is one or more IL- 18 variants.

[0176] In various embodiments, IL-15 variant, the IL- 18, the fragment of the IL-18, the IL-18 variant, or the fragment of the IL- 18 variant, and the immune checkpoint-taigeting fragment are each independently fused to the C- tcrminus of the scaffold polypeptide, hr various embodiments, (b) is one or more IL-18 variants.

[0177] In various embodiments, IL- 15 variant, tire IL- 18, the fragment of the IL- 18. the IL- 18 variant, or the fragment of the IL- 18 variant, and the immune checkpoint-targeting fragment are each independently fused to the N- terminus ofthe scaffold polypeptide. In various embodiments, (b) is one ormore IL-18 variants.

[0178] In various embodiments, the scaffold polypeptide is an antibody, and IL- 15 variant, the IL- 18, the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, and the receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or the one or more polypeptides capable of binding a tumor associated antigen (TAA), are each independentiy fused to any one of one of a C-terminus or an N-terminus of a heavy chain or a light chain, or fused to any one of a CH2 domain, or hinge region of the antibody.

[0179] In various embodiments, the scaffold polypeptide is an antibody, and the IL- 15 variant, the interleukin18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, are each independently fused to any one of one of a C-terminus or an N- terminus of a heavy chain or a light chain, or fused to any one of a CH2 domain, or hinge region of the antibody.

[0180] In various embodiments, the scaffold polypeptide is an antibody, and the IL- 15 variant, the interleukin18 (IL- 18), the fragment ofthe IL- 18, tire IL- 18 variant, or the fragment ofthe IL- 18 variant, or tire immune checkpointtargeting fragment, are each independently fused to any one of one of a C-terminus or an N-terminus of a heavy chain or a light chain, or fused to any one of a CH2 domain, or hinge region of tire antibody.

[0181] In various embodiments, the scaffold polypeptide is an Fc region, and the IL- 15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or the one or more polypeptides capable of binding a tumor associated antigen (TAA), are each independently fused to any one of one of a C-terminus or an N-terminus of tire Fc region.

[0182] In various embodiments, the scaffold polypeptide is an Fc region, and the IL- 15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor ty rosine kinase-like orphan receptor 1 (R0R1) VHH, are each independently fused to any one of one of a C-terminus or an N- terminus of the Fc region.

[0183] In various embodiments, the scaffold polypeptide is an Fc region, and the IL- 15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the immune checkpoint- targeting fragment, are each independentiy fused to any one of one of a C-terminus or an N-terminus of the Fc region.

[0184] In various embodiments, tire scaffold polypeptide is an antibody or a fragment thereof. In various embodiments, tire antibody is an IgA, IgM, IgG, or IgE antibody. Additional examples of antibodies include but are notlimited to monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, and single chain (ScFv). The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibodies are human or humanized antibodies.

[0185] In various embodiments, the antibody is an anti-PD-1 antibody or anti-PD-Ll antibody. Examples of anti-PD-1 antibodies include but are not limited to pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, and anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1). Examples of anti-PD-Ll antibodies include but arc not limited to garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053. envafolimab (KN035), MDX-1105, betifisoliniab (MSB2311), adebrelimab (SHR-1316). atezolizumab, avelumab, durvalumab, BMS-936559, CK-301. and M7824.

[0186] In various embodiments, two arms (or chains) of immunoglobulin heavy chain constant regions (e g.,Fc polypeptides) can be heterodimerized by creating “knobs-in-holes” (KiH) mutations in the CH3 domain. This structural feature in the polypeptide anns allows for assembly of tw o half antibodies (e.g., Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each comprising a CH3 domain, wherein tire polypeptides meet at an engineered interface within the CH3 domain, and the one polypeptide contains an engineered protuberance (“knob”) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and another polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue, hr some embodiments, the engineered interface of a heteromultimer includes at least two protuberance-into-cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia. J. Mol. Biol. 105: 1-14, 1975. For example, import residues for the formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, import residues for the formation of a cavity can be alanine (A), serine (S), threonine (T) and valine (V); and preferably the original residue for the fonnation of the cavity has a large side chain volume, such as ty rosine, arginine, pheny lalanine or tryptophan. For example, a T366W mutation in CH3 domain for the "knob' / protubcrance chain, and a T366S / L368A / Y407V mutation in CH3 domain for the “hole’Vcavity chain. Additionally, the KiH configuration may be coupled further mutations to permit S-S disulfide linkage between the two chains. In various aspects wherein the protein / polypeptide is a heterodimer of a the KiH configuration, the IL- 18 (or its fragment, variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) iminunoglobulin heavy chain constant regions / chains (i.e. knob or hole).

[0187] In various embodiments, the scaffold polypeptide is a fragment crystallizablc (Fc) region or a fragment thereof. In various embodiments, the fragment crystallizable (Fc) region is an Fc region from an IgG4, knobs-in-hole (KiH) Fc, or IgGl . In various embodiments, tire fragment cry stallizablc (Fc) region is a knobs-in-holc (KiH) Fc.

[0188] In various embodiments, tire Fc region is an Fc region from an IgG4. knobs-in-holc (KiH) Fc, or IgGl. In various embodiments, Fc region can be a human IgGl, IgG2, or IgG4.

[0189] In some embodiments, the two or more arms (or chains) of immunoglobulin heavy chain constant regions (e.g., Fc polypeptides) can contain another symmetric-to-asymmetric steric complementarity' design (e.g., HA- TF, ZW1), a charge-to-charge swap interaction (DD-KK), a charge-to-steric complementarity swap plus additional long- range electrostatic interaction (e.g., EW-RVT), or an isotype strand swap design (e.g.. strand-exchange engineered domain (SEED)), or Xrnab, 7.8.60, Electrostatic Steering, A107. or Duobody, so as to form heterodimeris / heteromultimers. Further description of these configurations and exemplary mutations / residues are seen in Front Immunol. 2016; 7: 394.

[0190] In various embodiments, the scaffold polypeptide is selected from fragment cry stallizablc (Fc) region, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof.

[0191] In various embodiments, the scaffold polypeptide is a polypeptide or protein capable of translocating into an endoplasmic reticulum (ER), or a fragment thereof.

[0192] Additional details and embodiments of the scaffold polypeptide are described herein and intended to be included as embodiments for these multi-cytokine fusion proteins.

[0193] In various embodiments, IL-15 variant comprises the sequence of Formula I:

[0194] Additional details and embodiments of the IL- 15 variant component of the fusion protein are described herein and intended to be included as embodiments for these multi-cytokine fusion proteins.

[0195] In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDS DVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDN KMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250) with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250. In various embodiments, the one to five amino acid substitutions is one amino acid substitution. In other embodiments, the one to five amino acid substitutions are two amino acid substitutions. In other embodiments, the one to five amino acid substitutions are three amino acid substitutions. In other embodiments, the one to five amino acid substitutions are four amino acid substitutions. In other embodiments, tire one to five amino acid substitutions are five amino acid substitutions, hr various embodiments, the IL- 18 variant comprises no more than five ammo acid substitutions, with tire exception of substituting cysteines. In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37- 193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDS DCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDN KMQFESSSYEGYFLAC EKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251.

[0196] In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDS DC RDNAPRTIFIISMYI<DSQPRGMAVTISVI<C EKISTLSCENKIISFKEMNPPDNIKDTI<SDIIFFQRSVPGHDN KMQFESSSYEGYFLAC EKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) with one or more amino acid substitutions at positions C74, C104, Cl 12, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149.

[0197] In various embodiments, the amino acid substitutions at one or more of C74, Cl 04, Cl 12, and C 164 are each independently substituted to valine, alanine or serine, hr various embodiments, the one to five amino acid substitutions arc one or more of: E42K, E42R, E42A, E42H, orE42Q; M87K, orM87H; K89G, K89A, or K89E; M96L, or M96I; or Ml 49V or Ml 491. In various embodiments, the one to five amino acid substitutions are E42K, E42R, E42A, E42H, or E42Q; M87K. or M87H; K89G, K89A, or K89E; M96L, or M96I; and M149V or M149I.

[0198] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 6B. In various embodiments, the fusion protein comprising the IL- 18 variant selected from Table 6B, further comprises a propeptide having an amino acid sequence selected from Propeptide column in Table 6B, and optionally from the same row as the IL- 18 variant. In various embodiments, the fusion protein comprising the IL- 18 variant selected from Table 6B and propeptide selected from Table 6B, further comprises a cleavage peptide selected from Table 6B, and optionally from the same row as the IL-18 variant and propeptide. As aparticular example, IEQD (SEQ ID NO:88) can be used.

[0199] In various embodiments, the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL- 18 variant further comprises its propeptide (PP) or a PP variant. In various embodiments, the PP or the PP variant is on the N-terminus end relative to the IL- 18, the fragment of IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant.

[0200] In various embodiments, the fusion protein further comprises one or more cleavage sites and the fusion protein is cleaved at the one or more cleavage sites by one or more proteases.

[0201] In various embodiments, the one or more cleavage sites is betw een the IL- 18, a fragment of IL- 18, anIL- 18 variant, or a fragment of the IL- 18 variant and the scaffold polypeptide, or within the PP, between PP or the PP variant and the IL- 18, a fragment of IL- 18, an IL- 18 variant, or a fragment of the IL- 18 variant, or within the PP, between the PP or the PP variant and the scaffold polypeptide, or within the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL- 18 variant, or within the PP, or a combination thereof.

[0202] Additional details and embodiments of the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL- 18 variant component of the fusion protein are described herein and intended to be included as embodiments for these multi-cytokine fusion proteins.

[0203] In various embodiments, the ROR1 VHH comprises: a polypeptide having SEQ ID NO:325 (complementarity -determining region (CDR) 1 of 2A11), a polypeptide having SEQ ID NO: 326 (CDR2 of 2A11), a polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination thereof; OR a variant of the polypeptide having SEQ ID NO:325 (CDRl of 2A11), a variant of the polypeptide having SEQ ID NO:326 (CDR2 of 2A11), a variant of the polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination tiiereof, wherein the variant of the polypeptide having SEQ ID NO: 325 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:325, wherein the variant of the polypeptide having SEQ ID NO:326 comprises one or more deletions, additions or a substitutions of an amino acid residue in the poly peptide having SEQ ID NO:326, wherein the variant of the polypeptide having SEQ ID NO:327 comprises one or more deletions, additions or asubstitutions of an amino acid residue in the polypeptide having SEQ ID NO:327, and wherein: the variant of the polypeptide having SEQ ID NO:325 and the variant of the polypeptide having SEQ ID NO:327 do not replace cysteine residues in the polypeptide having SEQ ID NO:325 and the polypeptide having SEQ ID NO:327, or the variant of the polypeptide having SEQ ID NO:325 and the variant of the polypeptide having SEQ ID NO:327 replaces one or both of the cysteine residues in the polypeptide having SEQ ID NO:325 and / or one or both of the cysteine residues in the polypeptide having SEQ ID NO:327 with an amino acid that contains a cross-linking functional group.

[0204] Additional details and embodiments of the R0R1 VHH component of the fusion protein are described herein and intended to be included as embodiments for these multi-cytokine fusion proteins.

[0205] In embodiments wherein the fusion protein comprises the scaffold polypeptide and (a) one or more of an interleukin 15 (IL-15) variant, and (b) one or more of an interleukin 18 (IL-18), a fragment of the IL-18. IL-18 variant, or a fragment of the IL-18 variant, the (a) one or more of an interleukin 15 (IL-15) variant, and (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant can be as described herein and intended to be included as embodiments for these multi -cytokine fusion proteins.

[0206] In embodiments wherein the fusion protein comprises tire scaffold polypeptide and (a) one or more of an interleukin 15 (IL-15) variant, and (b) one or more of an IL-18 variant, the (a) one or more of an interleukin 15 (IL-15) variant, and (b) one or more of an IL- 18 variant can be as described herein and intended to be included as embodiments for these multi-cytokine fusion proteins.

[0207] hr various embodiments, the fusion protein, comprising the scaffold polypeptide and (a) one or more of an interleukin 15 (IL- 15) variant, and (b) one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18. IL- 18 variant, or a fragment of tire IL-18 variant, is selected from Table 3A. For example, the fusion protein is FUSE-697, FUSE-916, FUSE-923, FUSE-943, FUSE-944, FUSE-1145, FUSE-1146, FUSE-1124 or FUSE 1136.

[0208] In various embodiments, the fusion protein, comprising the scaffold polypeptide and (a) one or more of an interleukin 15 (IL- 15) variant, and (b) one or more of air interleukin 18 (IL- 18), a fragment of tire IL- 18, IL- 18 variant, or a fragment of the IL- 18 variant, is encoded by a polynucleotide selected from Table 3B. For example, the fusion protein is FUSE-697, FUSE-916, FUSE-923, FUSE-943. FUSE-944, FUSE-1145, FUSE-1146, FUSE-1124 or FUSE 1136.Polynucleotides, expression vectors, cells and methods of producing fusion proteins.

[0209] Various embodiments provide for a polynucleotide encoding any one of tire fusion protein of the present invention as described herein.

[0210] Various embodiments provide for an expression vector comprising any one of the polynucleotide of the present invention as described herein.

[0211] Various embodiments provide for a cell transfected with any one of the expression vector of the present invention as described herein. In various embodiments, the cell is a mammalian cell. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, NSO cells (a mouse myeloma cell line), PER.C6® cells, and human embryonic kidney cells (HEK cells), In various embodiments, the cell is abacterial cell or yeast cell.

[0212] Various embodiments provide for a method of producing any one of the fusion proteins of the present invention described herein, comprising: culturing any one of the cells of the present invention described herein, in cell culture medium to allow the fusion protein to be produced, and optionally secreted into the cell culture medium. In various embodiments, the method further comprises isolating the fusion protein, In various embodiments, the method further comprises purifying the fusion protein.

[0213] In some embodiments, using a Chinese hamster ovary (CHO) expression system, the fusion protein is produced via a process including the steps of: ( 1 ) cell recovery, which may be to recover frozen CHO cells via water bath at 37°C; (2) cell subculturing, which may be to sub-culture the cells and adjust the cell density to 6x 106ml for transfection; (3) transfection and expression, using a solution 1 (in which a plasmid is diluted w ith a diluting agent), a solution 2 (in which a transection reagent is diluted with a / the diluting agent), and then mixing the solution 1, the solution 2 and the CHO cells, followed by incubating the mixture at a shaker for expression for 12-14 days at 32°C to collect the supernatant of the culture after centrifuge.

[0214] In some embodiments, a purification process is performed after the expression of the fusion protein. In some embodiments, a purification process includes the steps of: (1) washing a column with a binding buffer (10 times volume) at a flow' rate of 1 mL / min; (2) loading a fusion-protein-containing sample into the column at a flow' rate of 1 mL / min; (3) washing the column with 10x volumes of PBS buffer with a flow rate of 1 mL / min; (4) eluting the protein from the column with 40 mM sodium citrate (pH3.4); optionally the elution sample may be collected into tubes (Iml / min) and measured for optical density (OD) using NanoDrop at 280 nm; and (5) performing dialysis, e.g., against PBS buffer in a dialysis bag overnight.Methods of use

[0215] Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering any one of the fusion protein of the present invention described herein, to a subject in need thereof. Expansion of T cells, B cells or natural killer (NK) cells, does not require binding to the tumor associated antigen, such as ROR1. In various embodiments, the subject has cancer.

[0216] Various embodiments provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering any one of the fusion protein of the present invention described herein, to a subject in need thereof. In various embodiments, the disease or condition is cancer.

[0217] Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering at least two of the following to asubject in need thereof: (a) interleukin 15 (IL- 15) variant or IL- 15 fusion protein, (b) interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, a fragment of the IL- 18 variant, or an IL- 18 fusion protein, and (c) a multispecific antibody construct comprising a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, and a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally tire activation receptor comprises cluster of differentiation (CD) 3, CD16, NKp46, or NKG2D, and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp30, CD2, ICOS, 0X40, CD40L and CD40. In various embodiments, the subject has cancer. Expansion of T cells, B cells or natural killer (NK) cells, does not require binding to the tumor associated antigen, such as R0R1.

[0218] Various embodiments provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering at least two of the following to a subject in need thereof: (a) interleukin 15 (IL- 15) variant or IL- 15 fusion protein, (b) interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, a fragment of the IL- 18 variant, or an IL- 18 fusion protein, (c) a multispecific antibody construct comprising a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, and a receptor binding polypeptide capable of binding an activation receptor and / or a costinnilatory receptor expressed on an immune cell, wherein optionally tire activation receptor comprises cluster of differentiation (CD) 3, CD 16, NKp46, or NKG2D, and wherein optionally tire costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp30, CD2, ICOS, 0X40, CD40L and CD40, and (d) one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA). In various embodiments, the subject has cancer. Expansion of T cells, B cells or natural killer (NK) cells, does not require binding to the tumor associated antigen, such as R0R1.

[0219] A method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, administering at least two of the following to a subject in need thereof: (a) interleukin 15 (IL-15) variant or IL-15 fusion protein, (b) interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, a fragment of the IL- 18 variant, or an IL- 18 fusion protein, and (c) a multispecific antibody construct comprising a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, and a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatoiy receptor expressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD 16, NKp46, or NKG2D, and wherein optionally the costimulatoiy receptor comprises cluster of differentiation (CD) 137, CD28. DNAM-1, NKp30, CD2, ICOS, 0X40, CD40L and CD40. In various embodiments, the disease or condition is cancer.

[0220] A method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, administering at least two of tire following to a subject in need thereof: (a) interleukin 15 (IL-15) variant or IL-15 fusion protein, (b) interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, a fragment of tire IL-f8 variant, or an IL-18 fusion protein, (c) a multispecific antibody construct comprising a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, and a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatoiy receptorexpressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD 16, NKp46, or NKG2D, and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp30, CD2, ICOS, 0X40, CD40L and CD40, and (d) one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA). hr various embodiments, the disease or condition is cancer.

[0221] In some embodiments, the cancer is a R0R1 -expressing cancer, such as lung cancer, hematological cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cancer, uterine cancer, bladder cancer, kidney cancer, melanoma, thyroid cancer, myeloid leukemia, mantle cell lymphoma, or multiple myeloma. The R0R1 -expressing cancer can be a lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). The R0R1 -expressing cancer can be a hematological cancer, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse laige B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN). The R0R1 -expressing cancer can be breast cancer. The R0R1 -expressing cancer can be prostate cancer. The R0R1 -expressing cancer can be pancreatic cancer. The R0R1 -expressing cancer can be colon cancer. The R0R1 -expressing cancer can be ovarian cancer. The R0R1 -expressing cancer can be renal cancer. The ROR1- expressing cancer can be uterine cancer. The RORl-expressing cancer can be melanoma.IL-15 variant details and embodiments

[0222] Described herein arc IL- 15 variants that can be used in the multi-cytokine fusion proteins of the invention. Various embodiments provide for an interleukin 15 (IL- 15) variant comprising the sequence of Fonnula I:

[0224] In various embodiments, he IL-15 variant is a variant selected from Table 1A. In various embodiments, he IL-15 is a variant selected from Table 3A.

[0225] In various embodiments, he IL-15 variants are not IL-15 variants described in U.S. Patent Application Publ. No. US 2019 / 0263877, which is herein incorporated by reference.

[0226] Also described herein are fusion proteins comprising IL-15 variants and a scaffold polypeptide that can be used in various methods relating to administering separate fusions proteins described herein. Various embodiments of the invention provide for a fusion protein, comprising an IL- 15 variant as described herein; and a scaffold polypeptide. These IL-15 fusion proteins can be used in accordance with the methods of the present invention as described herein.

[0227] In various embodiments, the fusion protein comprises two or more IL- 15 variants as described herein; and a scaffold polypeptide. In various embodiments, the two or more IL-15 variants can be 3, 4, 5 or 6 IL-15 variants. In various embodiments, the tw o or more IL-15 variants can be up to 8 IL-15 variants.

[0228] hr various embodiments, the IL- 15 variant is fused to the C-terminus of the scaffold polypeptide. In other embodiments, the IL- 15 variant is fused to the N-terminus of the scaffold polypeptide.

[0229] In still other embodiments, wherein the scaffold polypeptide is an antibody, the IL- 15-variant is fused to any one of the C-terminus or N-terminus of the heavy chain or light chain, or fused to any one of the CH2 domain, or hinge region (e.g., between the CH2 and CHI domains).

[0230] In various embodiments, the scaffold polypeptide is an antibody or a fragment thereof. In various embodiments, the antibody is an IgA. IgM, IgG. or IgE antibody.

[0231] In various embodiments, the antibody is an anti-PD-1 antibody or anti-PD-Ll antibody. Examples of anti-PD-1 antibodies include but are not limited to pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, and anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1). Examples of anti-PD-Ll antibodies include but are not limited to garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824.

[0232] In various embodiments, tire scaffold polypeptide is a Fc region or a fragment thereof. In various embodiments, the scaffold polypeptide is a Fc region or a fragment thereof and tire scaffold polypeptide does not comprise a Fab.

[0233] In various embodiments, two arms (or chains) of immunoglobulin heavy chain constant regions (e g.,Fc polypeptides) can be heterodimerized by creating “knobs-in-holes” (KiH) mutations in the CH3 domain. This structural feature in the polypeptide arms allows for assembly of two half antibodies (e.g., Fc hctcrodimcr; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each comprising a CH3 domain, wherein tire polypeptides meet at an engineered interface within the CH3 domain, and the one polypeptide contains an engineered protuberance ('‘knob’’) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and another polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue, In some embodiments, the engineered interface of a heteromultimerincludes at least two protuberance-into-cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, import residues for the formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably the original residue for the fonnation of tire protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, import residues for the formation of a cavity can be alanine (A), serine (S). threonine (T) and valine (V); and preferably the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. For example, a T366W mutation in CH3 domain for the ‘'knob’7protuberance chain, and a T366S / L368 A / Y407V mutation in CH3 domain for tire “hole’Vcavity chain. Additionally, the KiH configuration may be coupled further mutations to pemiit S-S disulfide linkage betw een tire two chains. In various aspects wherein tire protein / polypeptide is a heterodimer of a the KiH configuration, tire IL- 18 (or its fragment, variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) immunoglobulin heavy chain constant regions / chains (i.e. knob or hole).

[0234] As such, in some embodiments, the Fc region is aknobs-in-hole (KiH) Fc.

[0235] In various embodiments, the IL-15 variant is fused to the knob of tire KiH Fc. In various embodiments, the IL-15 variant is fused to the hole of the KiH Fc.

[0236] In various embodiments, the Fc region is an Fc region from an IgG4. knobs-in-hole (KiH) Fc, or IgG In various embodiments, Fc region can be a human IgGl, IgG2, or IgG4.

[0237] In some embodiments, the two or more anns (or chains) of immunoglobulin heavy chain constai regions (e.g., Fc polypeptides) can contain another symmetnc-to-asymmctnc steric complementarity’ design (e.g., H7 TF. ZW1), a charge-to-charge swap interaction (DD-KK), a charge-to-steric complementarity swap plus additional loni range electrostatic interaction (eg., EW-RVT), or an isotype strand swap design (e.g.. strand-exchange engineers domain (SEED)), or Xmab, 7.8.60, Electrostatic Steering, A107, or Duobody, so as to fon heterodimeris / heteromultimers. Further description of these configurations and exemplary mutations / residues are seen i Front Immunol. 2016; 7: 394.

[0238] In various embodiments, the scaffold polypeptide of the fusion protein comprises a globular protei human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding doma: thereof, or a fragment thereof.

[0239] In various embodiments, one or more polypeptides can be inserted between the IL- 15 variant and d antibody of the IL-15 fusion proteins, In various embodiments, the polypeptide can be inserted or conjugated at the b tenninus. at the C-terminus, or both the N-terminus and C- terminus of the antibody. In various embodiments, ti polypeptide comprises apolypeptide linker conjugating the IL-15 variant and the antibody.

[0240] In various embodiments, one or more polypeptides can be inserted betw een the IL- 15 variant and the I region of the IL-15 fusion proteins. In various embodiments, the polypeptide can be inserted or conjugated at the bterminus, at the C-terminus, or both tire N-terminus and C- terminus of the Fc region. In various embodiments, the polypeptide comprises a polypeptide linker conjugating the IL- 5 variant and the Fc region, hr these embodiments, the fusion protein does not include Fab region of an antibody.

[0241] The antibodies that can be fused to IL- 15 can include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab. Fab', F(ab’)2. Fv. Fc, etc.), chimeric antibodies, bispecific antibodies, and single chain (ScFv). The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibodies are human or humanized antibodies.

[0242] In various embodiments, the IL- 15 fusion proteins are IL- 15 fusion proteins listed in Table 1A. That is, an IL- 15 fusion protein comprising polypeptide 1, polypeptide 2, and polypeptide 3.

[0243] In various embodiments, the IL- 15 fusion protein comprises polypeptide 1, polypeptide 2, and polypeptide 3 listed in Table 1 A, except the linker in polypeptide 2 is a different peptide linker. For example, the linker can be a flexible linker that is generally' about 10 to 25 amino acids in length. Additional examples of linkers include but are not limited to a dimer of two amino acids, a tri-mer of three amino acids, or a peptide selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX, (SEQ ID NO:236))n, and (X GGGG (SEQ ID NO:317))n wherein Xλ is Q, A, E or S and n=l-5 or an integer larger than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))n where n is an integer between 1 and 5. theret an amino acid linker of 25 amino acids or shorter in length.

[0244] In various embodiments, the IL- 15 fusion protein comprises an IL- 15 variant listed in Table 1A (Table 3B.Table 1A - Polypeptide sequences (IL-15 embodiments)Note: polypeptide 1 is the “hole” heavy chain; Polypeptide 2 is the “knob” heavy' chain, a linker (underlined) and an IL-15 variant; Polypeptide 3 is the light chain.IL-15 variant is also listed separately, but is included in Polypeptide 2.Table IB -Polypeptide sequences (IL-15 embodiments)Note: Polypeptide 1 is the heavy chain and polypeptide 2 while left blank is identical to Polypeptide 1. Polypeptide 3 is the light chain.Table 3A - Polypeptide sequences relating to IL15 / IL18 fusion proteinsNote: polypeptide 1 is the ‘hole” heavy chain; Poly peptide 2 is the “knob” heavy chain; Polypeptide 3 is the light chain. Mature IL18-variant and IL-15 variant are also listed separately, but is included in Polypeptide 1, 2 or 3.Table 3A.1 Polypeptide sequences relating to embodiments involving IL15 / IL18 fusion proteinsNote: polypeptide 1 is the “hole” heavy chain; Polypeptide 2 is the “knob” heavy chain; Polypeptide 3 is the light chain.

[0245] Various embodiments provide for a polynucleotide encoding an IL- 15 variant of the present invention described herein. For example, the nucleic acid sequences may encode in a 5’ to 3' direction, the IL- 15 variant or the IL- 15 fusion protein.

[0246] Various embodiments provide for a polynucleotide encoding an IL- 15 fusion protein of the present invention described herein. For example, the nucleic acid sequences may encode in a 5' to 3' direction, the IL- 15 variant.

[0247] As such, the polynucleotide encoding an IL- 15 fusion protein of the present invention comprises polynucleotide 1, polynucleotide 2 and polynucleotide 3 listed in Table 2A. In other embodiments, the polynucleotide encoding an IL- 15 fusion protein of the present invention comprises polynucleotide 1, polynucleotide 2 and polynucleotide 3 listed in Table 2A, except the nucleotides encoding the linker encodes a different linker.

[0248] For example, the linker can be a flexible linker that is generally about 10 to 25 amino acids in length.Additional examples of linkers include but arc not limited to a dimer of two amino acids, a tri-mcr of three amino acids, or a peptide selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX, (SEQ ID NO:236))n, and (X GGGG (SEQ ID NO:317))n wherein Xλ is Q, A. E or S and n=l-5 or an integer larger than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))n where n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in length. As such, the nucleotides encoding the linker in polynucleotide 2 will encode one of these aforementioned linkers instead.

[0249] Exemplary polynucleotides are in Table 2A.Table 2A Polynucleotides (IL-15 embodiments)Note: Polynucleotide 1 encodes the “hole” heavy chain; Polynucleotide 2 encodes the “knob” heavy chain, a linker, and an IL-15 variant, Poly nucleotide 3 encodes the light chain.Table 3B.1 Polynucleotide Sequences relating to embodiments involving IL15 / IL18 fusion proteinsSequences encoding the fusion proteins in Figure 12A and Figure 12BTable 4. Expression Yield of the proteinsIL-18 details and embodiments

[0250] The contents of International Application No. PCT / US2023 / 071663, filed August 4. 2023. entitled. ‘'IL-18 Fusion Proteins and Methods of Producing IL-18,” and International Application No. PCT / US2024 / 027248, filed May 1, 2024, entitled, “Methods of Using IL-18 Fusion Proteins” are incorporated by reference herein in their entirety as though fully set forth.

[0251] Described herein are IL- 18 variants that can be used in the multi-cytokine fusion proteins of tire invention. Described herein are also IL- 18 fusion proteins comprising IL- 18 variants that can be used in the methods relating to administering separate fusion proteins.

[0252] Various embodiments provide one or more fusion proteins, each comprising (i) an IL-18, a fragment of IL- 18, an IL- 18 variant, or a fragment of the IL- 18 variant, and (ii) a first protein capable of translocating into an endoplasmic reticulum (ER) including a cytosolic or nuclear protein engineered to translocate into an endoplasmic reticulum (ER) by means of addition of signal peptide / leader sequence onto tire N-tenninus of such an engineered protein, or a fragment of said proteins. Preferably, the protein capable of translocating into an ER has an amino acid sequence which initiates transport of a protein (e.g., the IL- 18 or its fragment, variant, or a fragment of its variant) across the membrane of the endoplasmic reticulum. In various embodiments, tire fusion protein comprises further an amino acid linker. For example, the amino acid linker can be between (a) the protein capable of translocating into an endoplasmic reticulum (ER) and (b) tire propeptide (or variant) or IL- 18 (or variant).

[0253] In some embodiments, the one or more fusion proteins do not comprise an IL-18 propeptide or its variant. An “IL-18 propeptide”, or “propeptide” or “PP” in this invention, may also be used interchangeably, which describes an amino acid sequence linked to IL- 18 or IL- 18 variant in an IL- 18 precursor or IL- 18 variant precursor, and which upon removal renders a mature IL- 18 or its fragment, or IL- 18 variant or its fragment thereof. For example, an IL- 18 propeptide may have a sequence of amino acid residues 1-36 of Uniprot ID Q14116.

[0254] In some embodiments, the one or more fusion proteins also include a propeptide (PP) or its variant.Examples of propeptide variants are provided herein, including those in Table 6B. This can inactivate IL- 18 or IL- 18 variant, and so a propeptide directly or indirectly is linked to the IL-18 or IL-18 variant fomrs a precursor IL-18 or precursor IL-18 variant. Preferably, The PP or its variant is on the N-terminus end relative to IL- 18 (or its fragment, variant, or a fragment of its variant) in the fusion protein. In some embodiments, the one or more fusion proteins also include a cleavage site, which is preferably based on a peptide substrate sensitive to enzymatic / protease cleavage. The cleavage site may be positioned within the PP, between the PP or its variant (if present) and the IL- 18 (or its fragment, variant, or a fragment of its variant); or may be positioned between the protein capable of translocating into an ER and the PP (if present); or may be positioned between the protein capable of translocating into an ER and the IL-18 or its fragment, variant, or a fragment of its variant, especially in the absence of a PP. In some embodiments, when PP is present, tlie cleavage site is positioned within the PP. In further embodiments, the one or more fusion proteins include (i) an IL- 18. a fragment of IL- 18. an IL- 18 variant, or a fragment of the IL- 18 variant, (ii), a propeptide (PP) or its variant, which inactivates IL- 18, and a cleavage site.

[0255] Examples of propeptide variants include a polypeptide havingAAEPVEDNXiINFVAMKFIDNTLYFIAEDDEN wherein Xi is any amino acid except cysteine (SEQ ID NO:238). In various embodiments, Xi is alanine, valine, isoleucine, leucin, metiiionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239). In various embodiments. Xi is valine (SEQ ID NO:78). In various embodiments, XI is serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, Xi is serine (SEQ ID NO:76).

[0256] In various embodiments, the fusion protein does not comprise a polypeptide consisting of the sequence X1-X2-X3-X+ between (i) the propeptide or propeptide variant, and (ii) the mature IL- 18 or mature IL- 18 variant, wherein Xi is L or absent, X2 is E or absent, X3 is S or absent, and X4 is D or absent.

[0257] In various aspects of the fusion proteins, the IL- 18 (or its fragment variant, or fragment of its variant) is linked by a polypeptide bond to the first protein capable of translocating into tire ER The fusion proteins may have a variety of configurations. Preferably, the N-terminus of tire IL-18 (or its fragment, variant, or a fragment of its variant) is linked by a polypeptide bond dirccdy or indircctiy to the C-tcrminus of tire first protein capable of translocating in the ER.

[0258] Yet in other embodiments, the C-terminus of the IL- 18 (or its fragment, variant, or a fragment of its variant) is linked by a polypeptide bond directiy or indirectly to the N-terminus of the first protein capable of translocating in the ER. As a nonlimiting example, the IL-18-variant (or IL-18, a fragment of IL-18, a fragment of theIL-18 variant) is fused to the N-terminus of a knob of a knob-into-hole heterodimeric IgGl protein with or without a propeptide (pp).

[0259] In further embodiments, where the C-tcrminus of the IL- 18 is linked to the N-tcnninus of tire first protein capable of translocating in (or through) the ER, a second protein capable of translocating through the ER is often fused to tire N-terminus of the IL- 18 to mediate masking. It is contemplated that a fusion protein further comprises (iii) a second protein capable of translocating into / through an ER, or a “scaffold” such as heat shock proteins (HSPs) that may not translocate through the ER. In some embodiments, if HSP (nuclear protein) or a cytosolic protein is fused to the N- terminus of tire IL- 18 to mediate masking, it often requires a signal peptide fused to the N-terminus of the “scaffold” to mediate transport to tire ER; and, if the scaffold is fused to the C-tcrminus of IL- 18 to serve to stabilize the complex, then a second protein capable of translocating through the ER is often fused to tire N-tenninus of the IL- 18 to mediate masking. Hence, in some embodiments, the IL- 18 (or its fragment, variant, or a fragment of its variant) is at the C- terminus of the fusion protein; in some embodiments, the N-tenninus of the IL- 18 (or its fragment, variant, or a fragment of its variant) is on the C-terminus end relative to the first protein capable of translocating in the ER, and the C-terminus of the IL- 18 (or its fragment, variant, or a fragment of its variant) is on the N-terminus end relative to tire second protein capable of translocating in the ER. The “first” or “second” protein capable of translocating in an ER is used as a relative reference. One or more exemplary amino acid sequences of each component of the fusion protein are shown in Tables 1 and 4.

[0260] Some embodiments provide that the first protein / polypeptide capable of translocating into an ER comprises an immunoglobulin heavy' chain constant region. In some embodiments, tire immunoglobulin heavy' chain constant region comprises an immunoglobulin heavy' chain constant region domain selected from tire group consisting of a CH2 domain, a CH3 domain, and a CH4 domain, or a combination thereof. In some embodiments, wherein the immunoglobulin heavy chain constant region comprises a CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin heavy chain constant region lacks at least a CHI domain. In some embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an immunoglobulin heavy' chain constant region present in the same species as the IL- 18. In other embodiments, the immunoglobulin heavy chain constant region is an immunoglobulin heavy chain constant region present in the same species as an organism with which a nucleic acid molecule encoding the fusion protein or a precursor of the fusion protein is transformed or transfected. Further embodiments provide that the fusion protein lacks an immunoglobulin variable domain (VH).

[0261] In various embodiments, the IL- 18 (or its fragment, variant, or fragment of its variant) is identical (in sequence) to that of a human origin, and the immunoglobulin heavy chain constant region comprises a hinge region, and a CH2 domain or a CH3 domain, and more preferably comprises a hinge region and bodi a CH2 domain and a CH3 domain.

[0262] In various embodiments, the IL- 18 (or its fragment, variant, or fragment of its variant) is at least 95%,90%, or 85% identical (in sequence) to that of a human origin, but with amino acid substitutions or other modifications that reduces affinity of the IL-18 (or its fragment, variant, or fragment of its variant) forIL-18BP. It is contemplated drat immunoglobulin heavy chain constant regions suitable for tire invention may be derived from immunoglobulins belonging to any of the five immunoglobulin classes referred to in the art as IgA (Iga), IgD (Igδ). IgE (Ige). IgG ( Igy ). and IgM (Igp). However, immunoglobulin heavy chain constant regions from the IgG class are preferred. Furthermore, the immunoglobulin heavy chain constant regions may be derived from any of the IgG antibody subclasses referred to in the art as IgGl, IgG2, IgG3, and IgG4. frnmunoglobulin heavy chain constant region domains have cross-homology among the immunoglobulin classes. For example, tire CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to tire CEB domain of IgM and IgE. Preferred immunoglobulin heavy chain constant regions include protein domains corresponding to a CH2 region and a CEB region of IgG, or functional portions or derivatives thereof. Further description of immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. No. 5,541,087, and U.S. Pat. No. 5,726,044, which are incorporated by reference herein.

[0263] In multiple embodiments, tire protein / polypeptide to be fused with the IL- 18 (or its fragment, variant, or a fragment of its variant) is a dimer of two immunoglobulin heavy chain constant regions / chains, optionally crosslinked by a pair of disulfide bonds between cysteines on adjacent hinge regions. In some embodiments, a hinge region may have an upper hinge domain, a core hinge domain, and a lower hinge domain. In some embodiments, an upper portion of the hinge domain may include or remove the cysteine that is known to form a disulfide bond with the light chain or a fab, resulting in sequences such as EPKSC (SEQ ID NO:241) or EPKSS (SEQ ID NO:242) or EPKSA (SEQ ID NO:243). For example, fusion proteins including IgGl-bascd ER translocating protein, except FUSE-501, FUSE-503, and FUSE-509, may have removed cysteine from the hinge region, e g., EPKSS (SEQ ID NO:242) in IgGl-based ER translocating protein, except for FUSE-507 (FUSE-507 has EPKSA (SEQ ID NO:243) in the hinge region). A hinge region may also contain a core hinge domain, such as comprising a sequence CPPCP (SEQ ID NO:244) or a variant where the cysteine is replaced. A hinge region may further include a lower hinge domain, such as comprising a sequence APELLGGP (SEQ ID NO:245) or APEAAGGP (SEQ ID NO:246). In another example, FUSE-509 has an IgG4-bascd ER-translocating protein, using a hinge region as depicted in Chiu ct al., Antibodies 2019, / >(4). 55, 2019. While constructs including immunoglobulin hinge regions are preferred, as depicted in the drawings, the invention contemplates that crosslinking at other positions may be chosen as desired. Furthermore, in some cases, two or more monomers may associate non-covalentiy to produce dimers or multimers. In various aspects wherein the protein / polypeptide is a dimer of two immunoglobulin heavy chain constant regions / chains, the IL- 18 (or its fragment, variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) immunoglobulin heavy chain constant regions / chains. In the case of a wild type IgG-Fc that fomis a homodimer, in various instances, a IL- 18 is placed on the C-temiinus of each monomer of the Fc, thereby having two IL- 18 placed on the C-temiinus of the Fc. In some instances, a heterodimer may form (e.g., in purification step) when one wild-type Fc fused to one IL-18 is mixed with another wild-type Fc not fused to IL- 18. In other aspects, an IL- 18 (or its fragment, variant, or a fragmentof its variant) is linked each of the two (or more) immunoglobulin heavy chain constant regions / chains in the fusion protein.

[0264] In some embodiments, tw o arms (or chains) of immunoglobulin heavy chain constant regions (c.g.,Fc polypeptides) can be heterodimerized by creating 'knobs-in-holcs" (KiH) mutations in the CH3 domain. This structural feature in the polypeptide arms allows for assembly of two half antibodies (e.g., Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise a first polypeptide and a second polypeptide each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within the CH3 domain, and the first polypeptide contains an engineered protuberance (“knob”) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and the second polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue. In some embodiments, tire engineered interface of a heteromultimer includes at least two protubcrancc-into-cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, import residues for the formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably tire original residue for the fonnation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, import residues for tire formation of a cavity can be alanine (A), serine (S). threonine (T) and valine (V): and preferably the original residue for the formation of the cavity has a laige side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. For example, a T366W mutation in CH3 domain for the “knob” / protuberance chain, and a T366S / L368A / Y407V mutation in CH3 domain for the “hole’ / cavity chain. Additionally, tire KiH configuration may be coupled further mutations to pennit S-S disulfide linkage between tire two chains. In various aspects wherein the protcin / polypcptidc is a heterodimer of a tire KiH configuration, tire IL-18 (or its fragment, variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) immunoglobulin heavy chain constant regions / chains (i.e. knob or hole).

[0265] In some embodiments, the two or more arms (or chains) of immunoglobulin heavy chain constant regions (c.g., Fc polypeptides) can contain another symmctnc-to-asymmctric stcric complementarity design (c.g., HA- TF, ZW1), a chaige-to-charge swap interaction (DD-KK), a chaige-to-steric complementarity swap plus additional long-range electrostatic interaction (e.g., EW-RVT), or an isotype strand swap design (e.g., strand-exchange engineered domain (SEED)), or Xmab, 7.8.60, Electrostatic Steering, A107, or Duobody, so as to form heterodimers / heteromultimens. Further description of these configurations and exemplary mutations / residues are seen in Front Immunol. 2016; 7: 394.

[0266] In additional embodiments, suitable proteins capable of translocating into the ER or a fragment thereof of the fusion protein is a globular protein, immunoglobular protein, or a fragment thereof. In various embodiments, suitable proteins capable of translocating into the ER or a fragment thereof of the fusion protein is a shortpolypeptide or protein engineered with a signal peptide for translocating into the ER. In some embodiments, suitable proteins capable of translocating into the ER or a fragment thereof of the fusion protein is a short polypeptide or protein being about 2 kDa or no greater than 250 kDa. As examples, the short polypeptide or protein is about 2-5 kDa, about 6- lOkDa, about 11-20 kDa, about 21-30 kDa, about 31-40 kDa, about 41-50 kDa, about 51-75 kDa, about 76-100 kDa, about 101-125 kDa, about 126-150 kDa, about 151-175 kDa, about 176-200 kDa, about 201-225 kDa, or about 256- 250 kDa. As further examples, the short polypeptide is 2, 3, or 4 amino acids, or flexible linkers or amino acid chain of 2-5, 6-10, 11-15, 16-20, 21-25, 26-30 amino acids. As further examples, the short polypeptide is 1, 2, 3, or 4 amino acids, or flexible linkers or amino acid chain of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acids. Additional examples of these short polypeptide include but are not limited to a dimer of two amino acids, a tri- mer of three amino acids, a 4-mer of four amino acids, a 5-mer of five amino acids, or a peptide selected from the group consisting of T. PT. MPT. S, GS, GGS, GGGS (SEQ ID NO:235). and (GGGGX, (SEQ ID NO:236))n wherein X, is Q. A, E or S and n=l-5 or an integer larger than 5. hr some embodiments, tire amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))n where n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in length. Additional examples include (X,GGGG (SEQ ID NO:317))n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GX GGG (SEQ ID NO:3 18))r, wherein X,. is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGX GG (SEQ ID NO:319))„ wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer laigerthan 5, (GGGX G (SEQ ID NO:320))n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. Still additional examples include (X, GGG)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GX,.GG)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGX,G)n wherein Xλ. is Q, A, E or S and n=l-5 or in some embodiments, an integer larger han 5, (GGGXQn wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer laiger than 5. Still additional examples include (X,GG)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. (GX,G)n wherein X;.. is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGX,)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments. Still additional examples include (X,G)n wherein Xλ is Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GX,.)n wherein Xλ is Q, A, E or S and n= 1 -5 or in some embodiments, an integer laiger than 5.

[0267] In additional embodiments, suitable proteins capable of translocating in an ER can be a globular protein, human serum albumin (HSA). beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof. Additional suitable proteins capable of translocating in an ER can include type I transmembrane proteins or a fragment thereof, or type II transmembrane proteins or a fragment thereof.

[0268] In various embodiments, the fusion protein comprising the short polypeptide or protein and the IL- 18 or IL- 18 variant, or fragments thereof, further comprises a second proteins capable of translocating into the ER or a fragment thereof. The second protein capable of translocating into the ER or a fragment thereof can be an Fc domain or HSA, beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chainvariable fragment (scFv), anticalm, designed ankynn repeat protein (DARPin), a binding domain thereof, and a fragment thereof, or type I transmembrane proteins or a fragment thereof, or type II transmembrane proteins or a fragment thereof as described herein. Figures 50A and 53A (first three from left to right) are nonlimiting examples of such fusion proteins.

[0269] In yet other embodiments, tire fusion protein further comprises a protein that cannot naturally translocate into the ER such as nuclear or cytosolic proteins fused to the N-terminus of IL- 18. For said proteins, a signal peptide (which may be termed a leader sequence), such as Ig-kappa leader sequence (e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO:247)) in FUSE-499, or one or more other signal peptides including but not limited to those derived from human albumin and human azurocidin, see Kober ct al. Biotechnol Bioeng. 2013 Apr; 110(4): 1164-73 is fused to the N-tenninus of the non ER translocating protein. For example, the signal peptide may be on the N-terminus end of the propeptide or of the IL- 18 (or its fragment, variant, or a variant of its fragment). A further example of a protein capable of translocating in / into / through ER may be a protein engineered with a signal peptide, e g., on the N-tenninus end. As an example, Hsp70 is a nuclear protein, but can be engineered to be an ER- translocating protein when fused or linked with a signal peptide on the N-terminus of Hsp70. In various embodiments, the addition of an N-tenninal signal peptide, such as the Ig-kappa leader sequence, is in place of a Fc, globular protein, or HSS that’ d otherwise be present in a fusion protein disclosed herein.

[0270] In some embodiments, the fusion protein (e.g.. a masked IL-18) further comprises a tumor taigeting fragment, e.g., a fragment that targets cell surface proteins including but not limited to a tumor associated antigen (TAA). For example, FUSE-517 as shown in FIG. 51G is a masked IL-18 fusion protein that also comprises an anti- EGFR antibody fragment, e.g., Fab of cetuximab. One or more antigen-targeting (preferably tumor antigen-targeting) fragments of known antibodies are conceived to be compatible with the fusion protein system disclosed herein.

[0271] In some embodiments, the fusion protein (e.g., a masked IL-18) comprises an activation receptor taigeting fragment, e g., a fragment that targets activation receptors on cell surface including but not limited to CD16 on natural killer cell surface. Activation receptor taigeting fragment can be one as described herein or one known in the art as of tire filing date of the present invention. Activation receptors include immunoreceptor tyrosine-based activation motif (ITAM)-associated receptors, such as CD 16 and NKp46. Activation receptors also include those participating in spontaneous NK cell activation, such as NKp46 (CD335), NKp30 (CD337), NKp44 (CD336), NKG2D (CD314). DNAM-1 (CD226). 2B4 (CD244). LFA-1 (CD1 la-CD18), and CD2. hr some embodiments, the fusion protein (e.g.. a masked IL- 18) comprises both an activation receptor targeting fragment and a tumor targeting fragment. Examples of anti-CD16 fragments include but are not limited to CH2 domains of IgGl, CH2 domain of IgG4. In some embodiments, the fusion protein (e.g., a masked IL-18) comprises a polypeptide fragment that targets an immune checkpoint, e g., fragment that targets an immune checkpoint expressed on T cell. For example, FUSE-694 as shown in FIG. 23 is a masked IL- 18 fusion protein that also comprises an anti-PDl fragment. Example immune checkpoints include but are not limited to PD-1, PD-LL CTLA-4, LAG-3. One or more immune checkpoint-targeting fragments ofknown antibodies are conceived to be compatible with the fusion protein system disclosed herein. Examples of anti- PD1 fragments include fragments (E.g. Fab, Fv) from pembrolizumab, nivolumab, pidilizumab, AMP-514, spartalizumab, cemiplimab, AK105, BCD-100, Bl 754091, JS001, LZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tislelizumab, or PF-06801591. Additional examples of anti-PDl fragments also include fragments (e.g.. Fab, Fv) from vopratelimab, camrelizumab, sintilimab, AMP-224, AMP-514, and Acrixolimab. Thus, for example, the fusion protein comprises an anti-PDf fragment from an anti-PDf antibody (e.g.. Fab, Fv), an IL-f8 variant, and a protein capable of translocating into an ER (e.g., a Fc domain), and optionally, a linker.

[0272] In some embodiments, the fusion protein (e.g., a masked IL-f8) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on the same surface as the IL-18 RC. Examples of such proteins include but are not limited to CD16, y9 TCR, 52 TCR or 51 TCR, NKp46, CD137, CD40 or NKG2D. Targeting polypeptide to CD16, v9 TCR, 52 TCR or 51 TCR, NKp46, CD137, CD40 orNKG2D can be one as described herein or one known in the art as of tire filing date of the present invention. In some embodiments, fusion protein (e.g., a masked IL- 18) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on a cell that does not contain an IL-18RC. In these embodiments, the IL-18 fusion protein will need to be delivered close to the IL-18R complex for a cis or density effect to result in the interaction between the IL- 18 fusion protein and the IL-18R complex. For example. TAA targeted IL- 18 fusion protein could get to interact with the IL-18R complex on T cells if (a) the fusion protein bridged T cells with TAA+ cells or (b) tire fusion protein was combined with another protein that bridged T cells with TAA+ cells or (c) the fusion protein bound to a TAA+ cell that naturally interacted with T cell via a secondary means (e.g. TCR / MHC interaction). In other examples, the fusion protein could be delivered to fibroblasts or other accessory cells in the tumor microenvironment and released by proteases such diat it can act on IL-18R+ T or NK cells at a distance.

[0273] Exemplary targeting polypeptides include those noted in Table 18 or fragments thereof. Of tiiose listed as the antigen-binding antibodies, tiieir VHH, Fab regions, or single-chain variable fragments (scFv) can be used as the antigen-binding site of the multispecific antibodies disclosed herein.

[0274] In some embodiments, enterokinase is used for site-specific cleavage of recombinant fusion proteins containing an accessible enterokinase recognition site. For example, enterokinase can specifically cleaves after the C- terminal end of the lysine residue at its cleavage site, Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 87). Therefore, the fragment produced from this cleavage reaction does not inherit any residues from the DDDDK (SEQ ID NO: 87) recognition sequence. Additionally, DDDDK (SEQ ID NO: 87) is a part of the octapeptide FLAG tag (DYKDDDDK (SEQ ID NO:248)), which can be utilized as a fusion tag for recognition by antibody, and for detection of fusion protein with Western blot analysis, as well as for purification of the fusion protein by Anti-FLAG affinity chromatography.

[0275] Preferably, a cleavage site can be based on peptide substrates sensitive to other enzymes, especially proteases highly expressed in tumor microenvironment, such as granzyme B, granzyme A, granzyme M, granzyme K, matrix metalloproteinase (MMP) 1 / 2 / 9 / 14 or other MMPs. Of note, granzymes are usually only upregulated in inflamedtumors. For example, a substrate sequence for granzyme B can be Ile-Glu-Xaa-Asp|Xaa-Gly (SEQ ID NO:249) with the cleavage at the Asp / Xaa peptide bond. Alternatively, a substrate sequence for granzyme B can also be Ile-Glu- Xaa-Aspj,, with the cleavage at the C-terminus end of Asp, and Xaa can be Gin (SEQ ID NO: 88) or another amino acid.

[0276] Several immune cells can release granzyme, such as T cells. NK cells, neutrophils, and mast cells. In several embodiments, a fusion protein comprising (a) a polypeptide fragment that targets an immune checkpoint expressed on an immune cell and / or a polypeptide fragment that targets an activation receptor on NK cell, and (b) a tumor targeting fragment, is effective for bringing the immune cell (e.g., T cell, NK cell) to the tumor, which can result in the release of granzymes that release IL- 18. For example, an IL- 18 fusion protein comprising a polypeptide fragment targeting an immune checkpoint protein can reverse exhaustion of NK and / or T cells that then are capable of releasing more granzymes.

[0277] In additional embodiments, the fusion protein comprises a cleavage site recognized by a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acidprotease, a metalloproteinase, a gelatinase, or an asparagine peptide lyase. In some embodiments, the protease cleavage site is recognized by a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, alrKl. a IrKlO, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisinlike protease, an actinidain. a bromelain, a calpain. a caspase, a caspase-3, a Mir 1 -CP, a papain, a HIV - 1 protease, a HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a legumain, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix mctalloprotcasc (MMP), a MMP1, a MMP2, a MMP3, a MMP8, a MMP9, a MMP10, a MMP11, a MMP12, a MMP13, a MMP14, an ADAM10, an ADAM17, an ADAM12, an urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific taiget (PSA, hK3), an interleukin- 1(3 converting enzyme, a thrombin, a FAP (FAP-a), a dipeptidyl peptidase, or dipeptidyl peptidase IV (DPPIV / CD26), a type II transmembrane serine protease (TTSP), a neutrophil elastase, a cathepsin G, a proteinase 3, a neutrophil serine protease 4, a mast cell chymase, a mast cell tryptase, a dipeptidyl peptidase, and a dipeptidyl peptidase IV (DPPIV / CD26). Nonlimiting examples of cleavage sites arc included in Tabic 6B. As a particular example, IEQD can be used.

[0278] It is contemplated that a variant, fragment, or a fiagment of a variant of the IL- 18 is suitable, and in some embodiments preferred, for the composition of the fusion protein. For example, a variant of mature IL- 18 can have one, two, three, four, five, or more amino acid substitutions compared to the wild type mature IL- 18. For example, one or more cysteines in tire IL- 18 or its propeptide are replaced with a natural or non-natural amino acid, such as from Cys to Ser, Ala, or Vai, so as to reduce aggregation of tire molecule in the fusion protein. Additional examples include cysteine to threonine, asparagine, or glutamine; cysteine to alanine, isoleucine, leucine methionine, phenylalanine, tyrosine, or tryptophan: cysteine to phenylalanine, alanine, aspartic acid, or asparagine; or cysteine to threonine, glutamine, aspartic acid, phenylalanine, isoleucine or histidine.

[0279] A variant of IL- 18 may have a 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65% or at least 60% sequence identity to wild type IL-18. In some embodiments, a variant of IL- 18 may have at least 60% and at most 83% sequence identity to wild type IL-18. In some embodiments, a variant of IL-18 may have between 60%-64%, 65%- 69%, 70%-74%, 75%-89%, or 80%-83% sequence identity to wild type IL-18. In some embodiments, the variant of IL- 18 in the fusion protein is released as an about 15 kDa functional fragment (e.g., on the electrophoresis gels as tested) when cleaved at a cleavage site of the fusion protein. (It is conceived that the released protein may be mature IL-18 which would normally run at 18 kDa but may appear as about 15 kDa due to the ladder being used or the specific polyacrylamide percentage in a gel.) A fragment of IL-18 may have a 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65%, or at least 60% sequence identity (and / or length) to wild type IL- 18. In some embodiments, an IL- 18 fragment produced by the fusion protein disclosed herein, especially after protease cleavage of the fusion protein, is less than 85% (e.g., about 83%, about 83%-80%, about 80%-75%, about 75%-70%, or about 70%-65%) in size compared to natural / wild- type mature IL-18; for example, an IL-18 fragment of about 15 kDa in size, preferably having comparable binding affinity for IL-18Ra / b as the wild type mature IL- 18, is fused to a propeptide (or PP variant) and an ER translocating protein (with or without mutations), and the fusion protein also includes a protease cleavage site, such drat upon protease cleavage, a small IL-18 fragment (e.g., about 15 kDa in size), is released. Preferably, this small IL-18 fragment maintains tire natural binding affinity for IL-18Ra / b and an equal or lower binding affinity relative to IL-18Ra / b for IL- 18BP. Preferably a variant, fragment, or a fragment of a variant of the IL-18 is capable of binding IL-18R and forming complex, so as to activate proinflammatory programs and / or NF-KB pathway. In some embodiments, the variant, fragment, or a fragment of a variant of the IL-18 is capable of having an increased binding affinity (e.g., 150%, 140%, 130%, 120%, 110%, or at least 100% relative to wild type IL-18) and / or inducing the biological activity of at least 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, compared to wild type IL-18. In some embodiments, the variant, fragment, or a fragment of a variant of tire IL- 18 is capable of having an increased binding affinity at 120%, 110%, or at least 100% relative to wild type IL-18, and / or inducing tire biological activity of at 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, compared to wrld type IL- 18. In additional embodiments, a variant, fragment, or a fragment of a variant of the IL- 18 has diminished binding to IL- 18 binding protein (IL-18BP), as compared to a w ild type IL-18.

[0280] In some embodiments, tire IL-18 or its fragment or variant cleaved from tire fusion protein has at least1,000, 2.000. 3,000, 5,000, 10,000, 30,000, 50,000, 70,000, 80,000, 90,000, or 100,000-fold increase in biological activity (e g., binding with IL-18R to form IL- 1 / IL- 1 R.a. / f> complex and induce downstream signaling), compared to an urrcleaved fomr in the fusion protein especially with propeptide. In further embodiments, the IL-18 or its fragment or variant cleaved from the fusion protein has a comparable biological activity, or within about 10, 20, 30, 40, or 50-fold difference irr tire biological activity, compared to recombinant human mature IL- 18.

[0281] In some embodiments, the IL- 18 or its fragment or variant cleaved from tire fusion protein has a binding affinity for its IL-18R complex with an equilibrium dissociation constant (KD) of about 18 nM, (e.g., 18 nM ±0.3 nM, 18 nM ± 0.5 nM, 18 nM ± 1.0 nM). In some embodiments, the IL-18 or its fragment or variant cleaved from the fusion protein has a binding affinity for its IL-18R complex which is about the same, or at least 100%, 95%, or 90%, compared to that of the wild type IL- 18. In some embodiments, the IL- 18 or its fragment or variant cleaved from the fusion protein has a binding affinity for its IL-18R complex w hich is greater dian that of the wild type IL-18, e.g., a binding affinity that is at least 105%, 110% compared to that of the wild type IL-18, or having a KD value at least 10% or 20% smaller than that of wild type IL-18. Preferably, the IL- 18 or its fragment or variant cleaved from the fusion protein has a reduced binding affinity for IL-18BP, compared to that of the wdd type IL-18. For example, in some instances, the IL- 18 or its fragment or variant cleaved from the fusion protein has a KD with IL-18BP of 18 nM or greater, such that it has a lower binding affinity to IL-18BP dian to IL-18R. In some instances, the IL- 18 or its fragment or variant cleaved from the fusion protein has a KD with IL-18BP of 18 nM or greater, whereas the wild type IL- 18 has a KD with IL-18BP of about 0.4 nM. It is also conceived diat KD may vary depending on instrument and protocol setup.

[0282] In various embodiments, the fusion protein comprises a first polypeptide or protein capable of translocating into an endoplasmic reticulum (ER) or a fragment thereof; and an interleukin 18 (IL- 18), a fragment of IL- 18, an IL- 18 variant, or a fragment of the IL- 18 variant, wherein the IL- 18, the fragment of IL- 18, the IL- 18 variant, or the fragment of the IL-18 variant is on the C-tenninus end of the fusion protein relative to the first polypeptide or protein capable of translocating into the ER. In various embodiments, the first polypeptide is not the wild-type IL- 18 propeptide. In various embodiments, the protein capable of translocating into an endoplasmic reticulum (ER) or a fragment thereof is not the wild-type IL- 18 propeptide. In addition to these features, additional features of the fusion protein are discussed herein.

[0283] In some embodiments, the fusion protein comprises an IL- 18 variant, In various embodiments, theIL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDVRDNAPRDFIISMYKDSQPRGMAVTISVKVEKISTLSVENKnSFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250) with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250). In various embodiments, the one to five amino acid substitutions is one amino acid substitution. In other embodiments, the one to five amino acid substitutions are two amino acid substitutions. In other embodiments, the one to five amino acid substitutions are three amino acid substitutions. In other embodiments, the one to five amino acid substitutions are four amino acid substitutions, In other embodiments, the one to five amino acid substitutions are five amino acid substitutions. In various embodiments, the IL- 18 variant comprises no more than five amino acid substitutions, with the exception of substituting cysteines. In various embodiments, the IL-18 variant has an amino acid sequence comprisingor consisting of amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDCRDNAPRIlFnSMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFrVQNED (SEQ ID NO:251) with one to five amino acid substitutions at positions E42, M87, K89. M96, and M149 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDCRDNAPRTIFnSMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).

[0284] In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDCRDNAPRTIFIISMYKDSQPRGMAV'TISVKC EKISTLSCENKnSFKEMNPPDMKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFrVQNED (SEQ ID NO:251) with one or more amino acid substitutions at positions C74, C 104, C 112, and C 164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149.

[0285] In various embodiments, the amino acid substitutions at one or more of C74, C104. Cl 12. and C164 are each independently substituted to valine, alanine or serine. In various embodiments, the one to five amino acid substitutions are one or more of: E42K, E42R, E42A, E42H, or E42Q; M87K, or M87H; K89G, K89A, or K89E; M96L, or M96I; or M149V or M149I. In various embodiments, the one to five amino acid substitutions are E42K, E42R, E42A, E42H, or E42Q; M87K, or M87H; K89G, K89A, or K89E; M96L, or M96I; and M149V or M149I.

[0286] In various embodiments, the fusion protein comprises an IL- 18 variant selected from Table 6B. In various embodiments, tire fusion protein comprising the IL- 18 variant selected from Table 6B, further comprises a propeptide having an amino acid sequence selected from Propeptide column in Table 6B, and optionally from the same row as the IL- 18 variant. In various embodiments, the fusion protein comprising the IL- 18 variant selected from Table 6B and propeptide selected from Table 6B, further comprises a cleavage peptide selected from Table 6B, and optionally from the same row as tire IL-18 variant and propeptide. As aparticular example, IEQD (SEQ ID NO:88) can be used.

[0287] In various embodiments, IL- 18 variant is an IL- 18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891, or PCT Publication No. WO 2022 / 038417, the IL- 18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though fidly set forth.

[0288] In various embodiments, the fusion protein further comprises a taigeting polypeptide. In some embodiments, the targeting polypeptide targets a protein on a cell surface, wherein the cell surface also has an IL- 18 RC or the cell is capable of expressing the IL- 18 RC. In various embodiments, the fusion protein binds to a cell having an IL-18 RC or capable of expressing the IL-18 RC upon activation of the cell, and activates the IL-18 RC signal.

[0289] In other embodiments, the targeting polypeptide targets a protein on a cell surface that does not have an IL- 18 RC or tire cell is not capable of expressing the IL- 18 RC. The cell not having the IL- 18 RC on its surface or not capable of expressing the IL- 18 RC is in close proximity to a cell expressing the IL- 18 RC or is capable of expressing the IL-18 RC. In other instances, the fusion protein can bring the cell not having the IL-18 RC on its surface or not capable of expressing the IL- 18 RC into close proximity to a cell expressing the IL- 18 RC or is capable of expressing the IL- 18 RC.

[0290] In various embodiments, the targeting polypeptide comprises a tumor associated antigen binding domain.

[0291] In various embodiments, tire fusion protein further comprises a binding domain for a protein expressed on immune cells, In various embodiments, the fusion protein further comprises a binding domain for a protein expressed on immune cells that express IL- 18 receptor complex or on immune cells that upon activation express the IL- 18 receptor complex.

[0292] In various embodiments, the fusion protein further comprises an antibody or antibody fragment. This enables the fusion protein to bind to a tumor cell, or to an immune cell or stromal cell in a tumor tissue, or to a tumor draining lymph node, or other secondary lymphoid organ. Examples of antibody fragments include Fc fragment, Fab fragment, Fv fragment, as well as others discussed herein.

[0293] hr various embodiments, the fusion protein further comprises a masking domain, hr some of these embodiments, the masking domain provides protection for IL- 18, and IL- 18 cannot be released by a protease, In some of these embodiments, a mature IL- 18 or mature IL- 18 variant can be released from a masking domain by a protease . In various embodiments, the protease is granzyme, which can be released from an immune cell. Examples of immune cells include but are not limited to an NK cell, a T cell, a neutrophil, or a mast cell. In various embodiments, the protease is a metalloprotease, which the metalloprotease can be expressed in a tumor microenvironment or tumor draining lymph node. Further examples of protease and types of granzymes are described herein. In various embodiments, the mature IL- 18 increases the activity of NK cells or T cells, and optionally the activity being one or more of proliferation, survival, and cytotoxicity.

[0294] In various embodiments, the fusion protein further comprises half-life extending molecule. A nonlimiting example of a half-life extending molecule is a half-life extending polypeptide; for example, human serum albumin (HSA) or an HSA-binding fragment. In various embodiments, the fusion protein has reduced activity as compared to wild-type IL-18 when not bound to a cell having the IL-18 RC. hr various embodiments, the reduced activity is at least a 75% reduction in activity as compared to wild-type IL-18.

[0295] In some embodiments, a mature IL- 18 variant on the C-tenninus of the mask is attenuated. For example, mature IL-18-mutl3A is 100 fold attenuated.

[0296] In various embodiments, the fusion protein comprises polypeptide 1 and polypeptide 2 selected fromTable 6A. In various embodiments, the fusion protein further comprises polypeptide 3 selected from Table 6A. Invarious embodiments, polypeptide 1 and polypeptide 2, and optionally polypeptide 3 is selected from the same row of Table 6A.

[0297] In various embodiments, the fusion protein comprises polypeptide 1 selected from Table 6A, wherein polypeptide 1 comprises HSA.

[0298] In various embodiments, the fusion protein does not comprise an IL- 18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891, or PCT Publication No. WO 2022 / 038417, the IL-18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though fully set forth.

[0299] Various embodiments of the invention include propeptide variants. In various embodiments, the propeptide variant has the following amino acid sequence: AAEPVEDNXiINFVAMKFIDNTLYFIAEDDEN wherein Xi is any ammo acid except cysteine (SEQ ID NO:238). In various embodiments, Xi is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239). In various embodiments, Xi is valine (SEQ ID NO:78). In various embodiments, XI is serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, Xi is serine (SEQ ID NO:76).

[0300] Various embodiments include for IL-18 variants to be used in the multi-cytokine fusion proteins of the present invention. In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of SEQ ID NO:250 with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDVRDNAPRTlFUSMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDnFFQRSVPGH DNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250). In various embodiments, the one to five amino acid substitutions is one amino acid substitution. In other embodiments, the one to five amino acid substitutions are two amino acid substitutions. In other embodiments, the one to five amino acid substitutions are three amino acid substitutions. In other embodiments, the one to five amino acid substitutions are four amino acid substitutions. In other embodiments, the one to five amino acid substitutions are five amino acid substitutions. In various embodiments, the IL- 18 variant comprises no more than five amino acid substitutions, with the exception of substituting cysteines.

[0301] In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 ofMAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD SDCRDNAPRTIFnSMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDlKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 ofMAAEPVEDNCINFVAMKHDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFnSMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).

[0302] In various embodiments, the IL- 18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of

[0304] hr various embodiments, the IL-18 variant is selected from the “Mature IL18 variant” column inTable 6B.

[0305] In various embodiments, the IL- 18 variant further comprises an IL- 18 propeptide or IL- 18 propeptide variant.

[0306] In various embodiments, the IL- 18 variant further comprises an IL- 18 propeptide variant having the following amino acid sequence: AAEPVEDNXiINFVAMKFIDNTEYFIAEDDEN wherein Xi is any amino acid except cysteine (SEQ ID NO:238). In various embodiments. Xi is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239). In various embodiments, Xi is valine (SEQ ID NO:78). In various embodiments, Xi is serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, Xi is serine (SEQ ID NO:76). In various embodiments, the IL-18 variant is selected from the "Mature IL18 variant” column of Table 6B, and further comprises a propeptide having an amino acid sequence selected from “Propeptide” column in Tabic 6B, and optionally from the same row as the IL-18 variant.

[0307] In various embodiments, the IL- 18 variant further comprises an IL- 18 propeptide variant, and a cleavage peptide. In various embodiments, the cleavage peptide selected from Table 6B. As a particular example. IEQD can be used.

[0308] In various embodiments, a fusion protein comprising an IL- 18 variant selected from Table 6B, a propeptide selected from Table 6B, and a cleavage peptide selected from Table 6B, and optionally from the same row as the IL- 18 variant and propeptide.

[0309] In various embodiments, the IL-18 variant is not an IL-18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891. or PCT Publication No. WO 2022 / 038417, the IL- 18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though frilly set forth.Table 6A. Amino acid sequences of respective components in exemplary fusion proteins (“FUSE”). Some of “Polypeptide 1” sequences, i.e., first polypeptide arm sequences, are identical in FUSE-422, FUSE-423, FUSE-424. FUSE-441. FUSE-442. FUSE-462, FUSE-480, FUSE-481. FUSE-484. FUSE-485, FUSE-486, FUSE-487, FUSE-499, FUSE-500, FUSE-505, FUSE-516, FUSE-517, FUSE-545, FUSE-546, FUSE547, FUSE5-556, FUSE-583-587,FUSE-599-602, FUSE-645, FUSE-686, FUSE-756-758, FUSE-775, FUSE-874-876, and FUSE-878-892.Fc hole; (4) Italicized and underlined font = Fc Knob; (5) bold font = FabPolypeptide 3 sequencesDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 73 )DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISS LQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTE ( SEQ ID NO : 74 )EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 75 )DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 280 )DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFT LKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ( SEQ ID NO : 281 )GGGGSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKIS TLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSI MFTVQNEDGGGGSGGGGSGGGGSGGGGSDIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSP QLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTVAAPSVFIFPPSD EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC ( SEQ ID NO : 282 )Table 6B. Sequences for the propeptide, cleavage and mature IL18 variant in Table 6A.Table 7. Nucleotide sequences encoding respective polypeptides of Table 6A.

[0310] Table 20. Yields of exemplary fusion proteins.

[0312] Table 5. Amino acid sequences for Fc heterodimerization including different versions of knobs-into-holes of respective polypeptide arms: for example, as scaffolding polypeptide or forming (part of) the protein capable of translocating to the ER, for exemplary fusion proteins.R0R1 details and embodiments

[0313] The contents of International Application No. PCT / US2023 / 070587, filed July 20, 2023, entitledReceptor Tyrosine Kinase-Like Orphan Receptor I (RORl)-Spccific VHH Antibodies and Multispccific Antibodies Thereof as Immune Cell Engagers are incorporated by reference herein as though fully disclosed.

[0314] Disclosed herein include antibodies (e.g., isolated antibodies), or antigen-binding fragments thereof, that immunospecifically bind to R0R1 that can be used with the mutli-cytokine fusion proteins of the present invention. Also described herein include multispecific antibody constmct that can be used in the methods involving administering separate fusion protein / constructs.

[0315] The inventors have identified human R0R1 -specific VHH produced in camels in response to stimulation by human R0R1, (thereby termed as VHH derived from Bactrian camels). These human RORl-stimulated. camel-derived VHH include:2A11 (a polypeptide comprising a polypeptide having an amino acid sequence of QVQLQESGGGSVPAGGSLRLSCAASGSTYSANCMGWFRQAPGKEREEVASMSIRSGRTYYSDSVKGRFTI SQDGSKNTLYLQLNSLKAEDTALYYCAAAYGGSRCVYNYRGQGTQVTVSS, SEQ ID NO: 331), and5A1 (a polypeptide comprising a polypeptide having an amino acid sequence of QVQLQESGGGSVQAGGSLKLSCTASGYTNRLKCMGWFRQAPGKEREEIAHSTGTGNTYYADSVKGRFTF SQDKVKNTVYLQMNTLKPDDTGMYYCAADVRPDGTTCHYNSGGQGTQVTVSS, SEQ ID NO:332).

[0316] An isolated anti-RORl (or R0R1 -specific) antibody, or RORl-binding fragment thereof, can comprise a VHH which comprises a polypeptide having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to the amino acid sequence of SEQ ID NO:331. In some embodiments, a VHH antibody (or nanobody) is provided, which can comprise, or consist of, the polypeptide having an amino acid sequence of SEQ ID NO:331.

[0317] In some embodiments, a polypeptide is provided comprising one or more complementaritydetermining regions (CDRs) for binding human R0R1, and these CDRs arc identified / prcsent in 2A11, wherein CDR1 of 2A11 consists of a polypeptide having an amino acid sequence of GSTYSANC (SEQ ID NO:325), CDR2 of 2A11 consists of a polypeptide having an amino acid sequence of MSIRSGRTY (SEQ ID NO:326). and CDR3 of 2A11 consists of a polypeptide having an amino acid sequence of AYGGSRCVYNY (SEQ ID NO:327).

[0318] In some embodiments, a polypeptide is provided comprising one or more of a variant of tire polypeptide having an amino acid sequence of SEQ ID NO:325, a variant of the polypeptide having an amino acid sequence of SEQ ID NO:326. and a variant of the polypeptide having an amino acid sequence of SEQ ID NO:327, wherein each of the variants comprises one or more deletions, additions, or substitutions of an amino acid residue compared to respective ”wild-type?’ polypeptides having tire amino acid sequence of SEQ ID NOs: 325. 326, or 327. respectively. In various embodiments, the substitutions are conservative substitutions. Preferably, a variant of the polypeptide having an amino acid sequence of SEQ ID NO:325, a variant of the polypeptide having an amino acidsequence of SEQ ID NO:326, and / or a variant of the polypeptide having an amino acid sequence of SEQ ID NO:327 are capable of binding human R0R1, more preferably still capable of immunospecifically binding human R0R1, or possess a binding affinity to R0R1 that is at least 90%, 80%, 70%, 60%, or 50% compared to respective "wild-type" CDR polypeptides present in 2A11. Preferably, one or both of the cysteine residues in the CDRs of 2A11 (i.e., cysteine residues in SEQ ID NO:325 and in SEQ ID NO:327) is unchanged, in a polypeptide that is a variant of 2A11 or in a polypeptide that is a variant of the polypeptide having an amino acid sequence of SEQ ID NO:325, 326. or 327. Hence, in some embodiments, a polypeptide comprising one or more of a variant of the polypeptide having an amino acid sequence of SEQ ID NO:325, a variant of the polypeptide having an amino acid sequence of SEQ ID NO:326, and a variant of the polypeptide having an amino acid sequence of SEQ ID NO:327, still maintains the cysteine residues as those in the polypeptide having an amino acid sequence of SEQ ID NO:325 and in tire polypeptide having an amino acid sequence of SEQ ID NO:327. Alternatively, a polypeptide can be a variant of 2A11. wherein one or both of tire cysteine residues in the CDRs of 2A11 is replaced with a natural or non-natural amino acid that contains a cross-linking functional group, so as to support formation of inter- / intra-molecular bonds: examples of these amino acids including but not limited to p-benzoyl-L-phenylalanine (Bpa), azide, and haloalkane. Preserving tire capability to form disulfide and / or intramolecular bonds at those residue locations where cysteine is present in tire CDR of 2A11 is indicated to be important, as data in FIG. 62G demonstrates that replacing tire cysteine residues with valine significantly weakens tire binding affinity of 2A11 to antigen.

[0319] In some embodiments, the amino acids in the non-CDR portion of 2A11 are modified, so as to generate a humanized version of 2A11 (comprising the CDRs of SEQ ID NOs: 325, 326, and 327 and a fiamework / non-CDR portion that is humanized), so as to better suit applications in human subjects. Therefore, various embodiments provide a humanized, anti-RORl antibody or fragment thereof.

[0320] In additional embodiments, an anti-RORl (or R0R1 -specific) heavy chain antibody is provided, which comprises ( 1) an antigen binding portion being a VHH comprising a polypeptide having an amino acid sequence of SEQ ID NO:331, or being a VHH comprising the polypeptides having amino acid sequences of SEQ ID NOs: 325, 326, and 327, and (2) a Fc-domain of an Ig heavy chain. Preferably, the heavy' chain antibody further comprises a hinge domain of an Ig heavy chain, so that the VHH is linked to the hinge and the Fc-domains of the Ig heavy chain. The disclosed anti-RORl heavy chain antibody, or an antigen-binding fragment thereof, include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin (Ig) stiucture, as well as the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk. In some embodiments, the anti-RORl heavy’ chain antibody comprises (1) an antigen binding portion being a VHH comprising a polypeptide having the amino acid sequence of SEQ ID NO:331, or a VHH comprising CDRs having amino acid sequences of SEQ ID NOs: 325, 326, and 327, and (2) the hinge and Fc-domains of an IgG heavy’ chain, wherein the IgG can be any of tire subclasses, IgGl, IgG2, IgG3, and IgG4.

[0321] An isolated anti-RORl (or ROR 1 -specific) antibody, or ROR1 -binding fragment thereof, can alternatively comprise a VHH comprising a polypeptide having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:331. In some embodiments, a VHH antibody (or nanobody) is provided, which can comprise, or consist of, a polypeptide having the amino acid sequence of SEQ ID NO:331.

[0322] In some embodiments, a polypeptide is provided comprising one or more complementaritydetermining regions (CDRs) of 5 Al, wherein CDR1 of 5A1 consists of a polypeptide having tire amino acid sequence of GYTNRLKC (SEQ ID NO:328), CDR2 of 5A1 consists of a polypeptide having the amino acid sequence of ISTGTGNTY (SEQ ID NO:329), and CDR3 of 5A1 consists of a polypeptide having the amino acid sequence of DVRPDGTTCHYNS (SEQ ID NO:330).

[0323] In some embodiments, a polypeptide is provided comprising one or more of a variant of the polypeptide having the amino acid sequence of SEQ ID NO:328, a variant of the polypeptide having the amino acid sequence of SEQ ID NO:329, and a variant of the polypeptide having tire amino acid sequence of SEQ ID NO:330, wherein each of the variants comprises one or more deletions, additions, or substitutions of an amino acid residue compared to respective '’w ild-type" polypeptide having tire amino acid sequences of SEQ ID NOs: 328, 329, or 330. In various embodiments, the substitutions are conservative substitutions. Preferably, a variant of the polypeptide having the amino acid sequence of SEQ ID NO:328, a variant of the polypeptide having the amino acid sequence of SEQ ID NO:329, and / or a variant of the polypeptide having the amino acid sequence of SEQ ID NO:330 are capable of binding human ROR1, more preferably still capable of immunospecifically binding human ROR1, or possess a binding affinity to human R0R1 that is at least 90%, 80%, 70%, 60%, or 50% compared to respective “wild-type” CDRs present in 5A1. Preferably, one or both of the cysteine residues in the CDRs of 5A1 (i.e., cysteine residues in SEQ ID NO:328 and in SEQ ID NO:330) are unchanged, in a polypeptide that is a variant of 5A1 or in a polypeptide that is a variant of tire polypeptide having an amino acid sequence of SEQ ID NO:328, 329, or 330. Hence, in some embodiments, a polypeptide comprising one or more of a variant of SEQ ID NO:328, a variant of SEQ ID NO:329, and a variant of SEQ ID NO:330 still maintains the cysteine residues as those in SEQ ID NO:328 and SEQ ID NO:330. Alternatively, a polypeptide can be a variant of 5A1, wherein one or both of the cysteine residues in the CDRs of 5 Al is replaced with a natural or non-natural amino acid that contains a cross-linking functional group, so as to support formation of inter- / intra-molecular bonds; examples of these amino acids including but not limited to p-benzoyl-L-phenylalanine (Bpa). azide, and haloalkane. Preserving the capability to form disulfide and / or intramolecular bonds at those residue locations is indicated to be important, as data in FIG. 62F demonstrates that replacing the cysteine residues with valine significantly weakens the binding affinity of 5A1 to antigen.

[0324] In some embodiments, the amino acids in the non-CDR portion of 5A1 are modified, so as to generate a humanized version of 5A1 (comprising the CDRs whose amino acid sequences are SEQ ID NOs: 328, 329.330, and a fiamework / non-CDR portion that is humanized), so as to better suit applications in human subjects. Exemplar}? humanized VHH derived from 5A1 include:5A1-H1 (a polypeptide having an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGYTNRLKCMGWFRQAPGKERELASISTGTGNTYYADSVKGRFTIS RDNSKNTLYLQMNSLKAEDTAVYYCAADVRPDGTTCHYNSRGQGTLVTVSS. SEQ ID NO:335),5A1-H2 (a polypeptide having an amino acid sequence of QVQLQESGGGLVQPGGSLRLSCTASGYTNRLKCMGWVRQAPGKEREEVATISTGTGNTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAMYYCA ADVRPDGTTCHYNSGGQGTQVTVSS, SEQ ID NO:336), 5A1-H3 (a polypeptide having an amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGYTNRLKCMGWFRQAPGKEREEVSTISTGTGNTYYADSVKGRFTI SQDKSKNTLYLRMNSLRAEDTALYYCAADVRPDGTFCHYNSGGQGTQVTVSS, SEQ ID NO:337),5A1-H4 (a polypeptide having an amino acid sequence of EVQLLESGGGLVPRGGSLRLSCTASGYTNRLKCMGWFRQAPGKEREEIATISTGTGNTYYADSVKGRFTIS RDNSRNTLYLQMKTLRAEDTAVYYCAADVRPDGTTCHYNSWGQGTQVTVSS, SEQ ID NO:338), and 5A1-H5 (a polypeptide having an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCTASGYTNRLKCMGWFRQAPGKEREEIATISTGTGNTYYADSVKGRFTF SRDNSKNTLYLQMNSLRAEDTAVYYCAADVRPDGTFCHYNSGGQGTQVTVSS, SEQ ID NO 339).

[0325] Additional exemplary humanized VHH are shown in Table 14.

[0326] In additional embodiments, an anti-RORI (or ROR1 -specific) heavy chain antibody is provided, which comprises (1) an antigen binding portion (i) being a VHH comprising a polypeptide having the amino acid sequence of SEQ ID NO: 332, or (ii) being a VHH comprising CDRs which are polypeptides having amino acid sequences of SEQ ID NOs: 328, 329, and 330. or (iii) being a VHH comprising a polypeptide having the amino acid sequence of any one of SEQ ID NO:335-339, or (iv) comprising one or more of (i), (ii) and (iii), and (2) a Fc-domain of an Ig heavy? chain. Preferably, the heavy chain antibody further comprises a hinge domain of an Ig heavy chain, so that the VHH is linked to the hinge and the Fc-domains of the Ig heavy? chain. The disclosed anti-RORI heavy? chain antibody, or an antigen-binding fragment thereof, include all isotypcs, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin (Ig) structure, as well as the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk. In some embodiments, tire anti-RORI heavy chain antibody comprises (1) an antigen binding portion being a VHH having the amino acid sequence of SEQ ID NO:332, or being a VHH comprising CDRs whose amino acid sequences are SEQ ID NOs: 328, 329, and 330, or being a VHH having the amino acid sequence of any? one of SEQ ID NOs:335-339, and (2) the hinge and Fc-domains of an IgG heavy? chain, wherein tire IgG can be any of tire subclasses, IgGl, IgG2, IgG3, and IgG4.

[0327] Polypeptides are also provided comprising ( 1) tw o or more repeats of a polypeptide having an amino acid sequence of SEQ ID NO:331. (2) two or more repeats of a polypeptide having an amino acid sequence of SEQ IDNO:332, (3) one or more repeats of a polypeptide having an amino acid sequence of SEQ ID NO:331 and one or more repeats of a polypeptide having an amino acid sequence of SEQ ID NO:332, (4) two or more repeats of a polypeptide having an amino acid sequence of any one of SEQ ID NOs:335-339, or (5) one or more repeats of a first polypeptide having an amino acid sequence of one of SEQ ID NOs:335-339 and one or more repeats of a second polypeptide having an amino acid sequence of one of SEQ ID NOs:335-339. where tire first and the second polypeptides have different amino acid sequences. In some embodiments, a polypeptide comprises a tandem sequence of a first VHH and a second VHH, wherein the first VHH and the second VHH can be the same or different. The amino acid sequence of the first VHH and the amino acid sequence of the second VHH can be individually selected from any one of SEQ ID NOs:331 and 332, or selected from any one of SEQ ID NOs:335-339. In one aspect, the first VHH and the second VHH are tire same and both have the amino acid sequence of SEQ ID NO:331; in another aspect, the first VHH and the second VHH are the same and both have the amino acid sequence of SEQ ID NO:332; in yet another aspect, the first VHH and the second VHH have the amino acid sequence of SEQ ID NO:331 and / or a variant of the polypeptide having an amino acid sequence of SEQ ID NO:331; in yet another aspect, the first VHH and the second VHH have the amino acid sequence of SEQ ID NO: 332 and / or a variant of the polypeptide having the amino acid sequence of SEQ ID NO:332; or in an alternative aspect, the first VHH may have the amino acid sequence of SEQ ID NO:331 or may be a variant of the polypeptide having the amino acid sequence of SEQ ID NO:331, and the second VHH may have the amino acid sequence of SEQ ID NO:332 or may be a variant of the polypeptide having the amino acid sequence of SEQ ID NO:332. In further embodiments, the polypeptide comprising the tandem sequence further comprises a linker, wherein the first VHH and the second VHH are connected with a linker. The linker may be on the N-terminal end of the VHH closer to the N-terminus of the Fc, or alternatively on the C-terminal end.

[0328] Various embodiments provide for a polypeptide, comprising: a polypeptide having a complementarity-determining region (CDR) 1. a polypeptide having a CDR2, and a polypeptide having a CDR3 selected from Table 15A, wherein CDR1, CDR2 and CDR3 are selected from the same row in Table 15A. Various embodiments provide for a polypeptide, comprising: a polypeptide having a complementarity-determining region (CDR) 1, a polypeptide having a CDR2, and a polypeptide having a CDR3 selected from Table 15B, wherein CDR1, CDR2 and CDR3 are selected from the same row in Table 15B. Various embodiments provide for a polypeptide, comprising: a variant of tire polypeptide having tire polypeptide having tire CDR1, the polypeptide having the CDR2, and the polypeptide having the CDR3 selected from Table 15 A, wherein the variant of the polypeptide having tire CDR1 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR1, wherein the variant of the polypeptide having the CDR2 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR2, and wherein the variant of the polypeptide having tire CDR3 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR3, wherein CDR1, CDR2 and CDR3 are selected from the same row in Table 15A. Various embodiments provide for a polypeptide, comprising: a variant of tire polypeptide having the polypeptide having the CDR1. the polypeptide having the CDR2. and the polypeptide having the CDR3 selected from Table 15B. whereinthe variant of the polypeptide having the CDR1 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR1, wherein the variant of the polypeptide having the CDR2 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR2, and wherein the variant of the polypeptide having the CDR3 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having the CDR3, wherein CDR1. CDR2 and CDR3 are selected from the same row in Table 15B. Various embodiments provide for a polypeptide, comprising: a polypeptide having a CDR1 of SEQ ID NO:328, a polypeptide having a CDR2 of SEQ ID NO:401, and a polypeptide having a CDR3 of SEQ ID NO:330. Various embodiments provide for a polypeptide, comprising: a polypeptide having a CDR1 of SEQ ID NO:397, a polypeptide having a CDR2 of SEQ ID NO:401, and a polypeptide having a CDR3 of SEQ ID NO:408. Various embodiments provide for a polypeptide, comprising: a polypeptide having a CDR1 of SEQ ID NO:397, a polypeptide having a CDR2 of SEQ ID NO:401, and a polypeptide having a CDR3 of SEQ ID NO:409. Various embodiments provide for a polypeptide, comprising: a polypeptide having a CDR1 of SEQ ID NO:397. a polypeptide having a CDR2 of SEQ ID NO:329, and a polypeptide having a CDR3 of SEQ ID NO:408. Various embodiments provide for a polypeptide, comprising: a polypeptide having a CDR1 of SEQ ID NO:397, a polypeptide having a CDR2 of SEQ ID NO:329, and a polypeptide having a CDR3 of SEQ ID NO:409. In various embodiments, the substitutions are conservative substitutions. Preferably , variants of the CDR1, CDR2 or CDR3 are capable of binding human R0R1, more preferably still capable of immunospecifically binding human R0R1, or possess a binding affinity to R0R1 that is at least 90%. 80%. 70%, 60%, or 50% compared to respective CDR polypeptides. In various embodiments, the polypeptide further comprises framewoik region (FWR) 1, framework region 2, framework region 3, and framework region 4 selected from Table 15B, and wherein FWR1, FWR2, FWR3, and FWR4 are selected from the same row in Table 15B In various embodiments, the polypeptide having CDR1, CDR2, CDR3, further comprises a FWR1, FWR2, FWR3 and FWR4 from a human IgG. In various embodiments, the polypeptide having a variant of CDRL a variant of CDR2, a variant of CDR3, further comprises a FWRL FWR2, FWR3 and FWR4 from a human IgG. In various embodiments, the polypeptide is a VEH polypeptide selected from Table 14. In various embodiments, the polypeptide is a variant of a VHH polypeptide selected from Table 14, wherein the variant comprises one or more deletions, additions or a substitutions of an amino acid residues of the polypeptide. In various embodiments, the variant comprises up to 5 deletions, additions or a substitutions of an amino acid residues of the polypeptide. In various embodiments, the variant comprises up to 1, 2 3 or 4 deletions, additions or a substitutions of an amino acid residues of the polypeptide... In various embodiments, the variant is at least 95% identical to tire polypeptide selected from Table 14. In various embodiments, the variant is at least 96%, 97%, 98%. or 99% identical to the polypeptide selected from Table 14. In various embodiments, the variant is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to its corresponding polypeptide selected from Table 14. In various embodiments, the polypeptide is a VHH polypeptide having an amino acid sequence of SEQ ID NO:340, SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364. hr various embodiments, tire polypeptide is a variant of a VHH polypeptide having an amino acid sequence of SEQ ID NO:340, SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364, wherein the variant comprises one or more deletions, additions or asubstitutions of an amino acid residues of the polypeptide. In various embodiments, the variant comprises up to 5 deletions, additions or a substitutions of an amino acid residues of the polypeptide. In various embodiments, the variant comprises up to 1, 2 3 or 4 deletions, additions or a substitutions of an amino acid residues of the polypeptide... In various embodiments, tire variant is at least 95% identical to tire VHH polypeptide having an amino acid sequence of SEQ ID NO:340. SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364. hr various embodiments, tire variant is at least 96%, 97%, 98%, or 99% identical to the VEIH polypeptide having an amino acid sequence of SEQ ID NO:340. SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364. In various embodiments, the variant is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to its VHH polypeptide having an amino acid sequence of SEQ ID NO:340, SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364...

[0329] The isolated antibody, or antigen-binding fragment thereof, can preferably bind to a region of tireR0R1 extracellular domain, and therefore bind to cells with surface expression of R0R1.

[0330] The anti-RORl VHH (nanobody), heavy chain antibodies, and / or antigen-binding fragments thereof, or polypeptides containing the anti-RORl VHH or CDRs thereof, as disclosed above, may be derived from any species by recombinant means. For example, the VHH, heavy' chain antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. Particularly, the VHH, heavy chain antibodies or antigen-binding fragments may be camelid or humanized version thereof. For use in administration to humans, non-human derived antibodies or antigen-binding fragments may be genetically or structural ly altered to be less antigenic upon administration to the human patient.

[0331] Also provided are polynucleotide sequences encoding the disclosed anti-RORl VHH, the disclosed anti-RORl heavy chain antibodies, and / or the disclosed polypeptides containing tire anti-RORl VHH or CDRs thereof. For example, vectors encoding the sequence of 2A 11 , 5 A 1 , or humanized versions of 2A 11 or 5 A 1 can be expressed in various transfection-compatible cell types, not limited to camel cells or human cells. Also, vectors encoding any one of the sequences noted in Table 14 can be expressed in various transfection-compatible cell types, not limited to camel cells or human cells. Vectors comprising the poly nucleotides are also provided. The vectors can be expression vectors, such as recombinant expression vectors. The expression vector may contain one or more additional sequences such as, but not limited, to regulatory sequences (e.g., promoter, enhancer), selection markers, and polyadenylation signals. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids. cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs). yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Also provided are cells expressing, and capable of expressing, the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). The disclosed antibodies may also be produced by hybridoma cells.Multispecific Antibodies and Multispecific Antigen-Binding Fragments

[0332] Also disclosed herein are multispecific (including bispecific or trispecific) antibodies, or multispecific antigen-binding fragments thereof, which bind to at least R0R1 and CD3 (anti-RORl / anti-CD3 antibodies), or bind to at least R0R1 and an activation receptor expressed on an immune cell (including but not limited to CD3, CD16, y9 TCR, 82 TCR or 81 TCR, NKp46, and NKG2D). In various embodiments, isolated bispecific antibodies, or bispecific antigen-binding fragments thereof, are provided which bind to R0R1 and CD3 (anti- RORl / anti-CD3 bispecific antibodies).

[0333] In various embodiments, the anti-RORl / anti-CD3 antibodies have at least a first antigen-binding site that immunospecifically binds R0R1 (ROR1 ann) and a second antigen-binding site that immunospecifically binds CD3 (CD3 ann). Exemplary anti-RORl / anti-CD3 antibodies of these embodiments are shown in FIG. 58A and 58E.

[0334] In some embodiments, the anti-RORl / anti-CD3 antibodies have at least a first arm that is R0R1- specific and a second arm that is bispecific, wherein the first arm that is ROR1 -specific comprises a first antigenbinding site drat immunospecifically binds R0R1, and the second arm that is bispecific comprises an antigen-biding site that immunospecifically binds CD3 and a second antigen-binding site that immunospecifically binds ROR1. Exemplary anti-RORl / anti-CD3 antibodies of these embodiments are shown in FIG. 58B and 58C.

[0335] hr various embodiments, the anti-RORl / anti-NKG2D antibodies have at least a first antigen-binding site that immunospecifically binds ROR1 (ROR 1 arm) and a second antigen-binding site that immunospecifically binds NKG2D (NKG2D arm).

[0336] In some embodiments, the anti-RORl / anti-NKG2D antibodies have at least a first arm that isR0R1 -specific and a second arm that is bispecific, wherein the first ann that is R0R1 -specific comprises a first antigenbinding site that immunospecifically binds ROR1. and the second arm that is bispecific comprises an antigen-biding site that immunospecifically binds NKG2D and a second antigen-binding site that immunospecifically binds ROR1.

[0337] In various embodiments, the anti-RORl / anti-CD16 antibodies have at least a first antigen-binding site that immunospecifically binds ROR1 (ROR1 ann) and a second antigen-binding site that immunospecifically binds CD16 (CD16 arm).

[0338] In some embodiments, the anti-RORl / anti-CD16 antibodies have at least a first arm that is ROR1- specific and a second arm that is bispecific, wherein the first arm that is ROR1 -specific comprises a first antigenbinding site that immunospecifically binds ROR1, and the second arm that is bispecific comprises an antigen-biding site that immunospecifically binds CD 16 and a second antigen-binding site that immunospecifically binds ROR1.

[0339] In various embodiments, the anti-RORl / anti-NKp46 antibodies have at least a first antigen-binding site that immunospecifically binds ROR1 (ROR1 arm) and a second antigen-binding site that immunospecifically binds NKp46 (NKp46 arm).

[0340] In some embodiments, the anti-RORl / anti-NKp46 antibodies have at least a first arm drat is ROR1- specific and a second ami that is bispecific, wherein the first ann that is ROR1 -specific comprises a first antigen-binding site that innnunospecifically binds R0R1, and the second arm that is bispecific comprises an antigen-biding site that immunospecifically binds NKp46 and a second antigen-binding site that immunospecifically binds R0R1.

[0341] In further embodiments, isolated multispccific antibodies, ormultispccific antigen-binding fragments thereof, are provided, which contains at least one RORl-binding site, at least one CD3-binding site, and a Fc-domain of an Ig heavy chain, and optionally a hinge domain of the Ig heavy chain. The Ig heavy chain can be any one of isotypes IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin (Ig) structure, as well as tire IgY iso type generally found in hen or turkey serum and hen or turkey egg yolk. Preferably, the Ig heavy chain is a IgG heavy chain, which can be any of tire subclasses, IgGl, IgG2, IgG3, and IgG4.

[0342] Hence, in some embodiments, anti-RORl / anti-CD3 antibodies have at least (1) a first antigenbinding site specific for R0R1, (2) a second antigen-binding site at least specific for CD3, or a bispecific antigenbinding site for ROR1 and CD3. and (3) the hinge and Fc domains of tw o or more chains of Ig, which can be dimerized or multimerized, wherein at least one heaving chain of the Ig may further comprise an antigen-binding site specific for R0R1, preferably on the C-terminus of the Ig heavy chain.

[0343] Hence, in other embodiments, anti-RORl / anti-NKG2D antibodies have at least (1) a first antigenbinding site specific for R0R1, (2) a second antigen-binding site at least specific for NKG2D, or a bispecific antigenbinding site for ROR1 and NKG2D, and (3) tire hinge and Fc domains of two or more chains of Ig, which can be dimerized or multimerized, wherein at least one heaving chain of the Ig may further comprise an antigen-binding site specific for ROR1 , preferably on the C-terminus of tire Ig heavy chain.

[0344] Hence, in other embodiments, anti-RORl / anti-CD16 antibodies have at least (1) a first antigenbinding site specific for ROR1, (2) a second antigen-binding site at least specific for CD 16 , or a bispecific antigenbinding site for R0R1 and CD16 , and (3) the hinge and Fc domains of two or more chains of Ig. which can be dimerized or multimerized, wherein at least one heaving chain of the Ig may further comprise an antigen-binding site specific for ROR1 , preferably on the C-terminus of fire Ig heavy chain.

[0345] Hcncc, in other embodiments, anti-RORl / anti-NKp46 antibodies have at least (1) a first antigenbinding site specific for R0R1, (2) a second antigen-binding site at least specific for NKp46 , or a bispecific antigenbinding site for ROR1 and NKp46 , and (3) the hinge and Fc domains of two or more chains of Ig. which can be dimerized or multimerized, wherein at least one heaving chain of the Ig may further comprise an antigen-binding site specific for ROR1 , preferably on the C-terminus of tire Ig heavy chain.

[0346] Alternatively, isolated multispecific antibodies, or multispecific antigen-binding fragments thereof, are provided, which contains at least one RORl-binding site, at least one CD3-binding site, and a scaffold protein such as human serum albumin. Alternatively, isolated multispecific antibodies, or multispecific antigen-binding fragments thereof, are provided, which contains at least one RORl-binding site, at least one NKG2D-binding site, and a scaffold protein such as human serum albumin. Alternatively, isolated multispecific antibodies, or multispecific antigen-binding fragments thereof, are provided, which contains at least one RORl-binding site, at least one CD16-binding site, and ascaffold protein such as human serum albumin. Alternatively, isolated multispecific antibodies, or multispecific antigenbinding fragments thereof, are provided, which contains at least one R0R1 -binding site, at least one NKp46 -binding site, and a scaffold protein such as human serum albumin.

[0347] The isolated multispecific antibodies, or multispecific antigen-binding fragments thereof, can comprise: a) a first antigen-binding site that immunospecifically binds RORL the first antigen-binding site comprising one or more of:2A 11 (the peptide having an amino acid sequence of SEQ ID NO: 331 ), a variant of 2A11 disclosed above,5A1 (the peptide having an amino acid sequence of SEQ ID NO:332), a variant of 5 Al disclosed above (such as a polypeptide having an amino acid sequence of any one of SEQ ID NOs:335-339), three, two, or at least one of the CDR1 (having an amino acid sequence of SEQ ID NO:325), CDR2 (having an amino acid sequence of SEQ ID NO:326), and CDR3 (having an amino acid sequence of SEQ ID NO:327) of 2A11, one or more variants of the three, two, or at least one of the CDR1 (having an amino acid sequence of SEQ ID NO:325), CDR2 (having an amino acid sequence of SEQ ID NO:326), and CDR3 (having an amino acid sequence of SEQ ID NO:327) of2Al l, three, two, or at least one of the CDR1 (having an amino acid sequence of SEQ ID NO:328), CDR2 (having an amino acid sequence of SEQ ID NO:329), and CDR3 (having an amino acid sequence of SEQ ID NO:330) of 5A1, and one or more variants of the three, two, or at least one of the CDR1 (having an amino acid sequence of SEQ ID NO:328), CDR2 (having an amino acid sequence of SEQ ID NO:329), and CDR3 (having an ammo acid sequence of SEQ ID NO:330) of 5A1; and b) a second antigen-binding site that immunospecifically binds CD3, CD16, NKp46, or NKG2D.

[0348] In various embodiments, the second antigen-binding site immunospecifically binds to CD3. In various embodiments, the antigen-binding site that immunospecifically binds to NKG2D.

[0349] In other embodiments, the isolated multispecific antibodies, or multispecific antigen-binding fragments thereof, can comprise: al) a first antigen-binding site comprising a polypeptide selected from Table 14, OR a2) a first antigen-binding site comprising CDR1, CDR2, and CDR3 selected from Table 15 A, OR a3) a first antigen-binding site comprising CDR1, CDR2, and CDR3 selected from Table 15B; and b) a second antigen-binding site that immunospecifically binds to CD3, CD16,NKp46, orNKG2D.

[0350] In various embodiments, the second antigen-binding site immunospecifically binds to CD3. In various embodiments, the antigen-binding site that immunospecifically binds to NKG2D.

[0351] In other embodiments, tire isolated multispecific antibodies, or multispecific antigen-bindingAugments thereof, can comprise: al) a first antigen-binding site comprising a variant of CDR1, a variant of CDR2, and a variant of CDR3 selected from Table 15 A, OR a2) a first antigen-binding site comprising a variant of CDR1, a variant of CDR2, and a variant of CDR3 selected from Table 15B; and b) a second antigen-binding site that immunospecifically binds to CD3, CD16, NKp46, orNKG2D.

[0352] In various embodiments, tire multispecific antibody comprises two or more VHH domains, or two or more single-chain variable fragments (scFv), capable of binding a tumor-associated antigen (TAA), wherein the two or more VHH domains are each independently tire one or more first polypeptides, wherein the one or more first polypeptides comprise a polypeptide selected from Table 14.

[0353] In various embodiments, the multispecific antibody comprises two or more VHH domains, or tw o or more single-chain variable fragments (scFv), capable of binding a tumor-associated antigen (TAA), wherein the two or more VHH domains are each independently the one or more first polypeptides, wherein tire one or more first polypeptides comprise a polypeptide having a sequence as set forth in SEQ ID NO:335-339.

[0354] In various embodiments, the multispecific antibody comprises two or more VHH domains, or tw o or more single-chain variable fragments (scFv), capable of binding a tumor-associated antigen (TAA), wherein the tw o or more VHH domains are each independently the one or more first polypeptides, wherein tire one or more first polypeptides comprise a polypeptide having a sequence as set forth in SEQ ID NO:335, 338-394.

[0355] Variants of CDR1, CDR2, and CDR3 are as discussed herein. In various embodiments, the first antigen-binding site further comprises a FWR1. FWR2. FWR3 and FWR4 selected from Table 15B. In various embodiments, the first antigen-binding site further comprises a FWR 1 , FWR2, FWR3 and FWR4 from a human IgG. In various embodiments, the second antigen-binding site immunospecifically binds to CD3. hr vanous embodiments, the antigen-binding site that immunospecifically binds to NKG2D.

[0356] Suitable antigen-binding sites can be in the form of a VHH, scFv, Fab, (Fab')i. one or more CDRs, or a fusion (tandem sequence optionally connected via a linker) of one or more of the VHH, scFv, Fab, (Fab )?. or CDRs. Suitable antigen-binding sites that immunospecifically bind to ROR1 include any of the above disclosed anti- ROR1 antibodies or ROR1 -binding fragments thereof. Suitable antigen-binding sites that immunospecifically bind to CD3 can be derived from CD3 antibodies disclosed in one or more publications including but not limited to U.S. Pat. No. 8,236,308, U.S. Patent App. Pub. Nos. 2010 / 0260668, 2013 / 0018174, 2012 / 0321626, 2013 / 0060011, 2013 / 0058936, 2013 / 0078249. and 2013 / 0058937.

[0357] In some embodiments, the antigen-binding site of the multispecific antibodies disclosed herein is connected to an Ig heavy chain constant region that contains a mutation. In some aspects, ‘’knob-in-hole” (KiH) mutations are present in tire CH3 domains of two arms (or chains) of Ig heavy' chain constant regions, so that tire twocan heterodimerize. This structural feature in the polypeptide arms allows for assembly of two half antibodies (e.g., Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise a first polypeptide and a second polypeptide each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within the CH3 domain, and tire first polypeptide contains an engineered protuberance (“knob”) in tire interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and the second polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue. In some embodiments, the engineered interface of a heteromultimer includes at least two protuberance-into- cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, import residues for tire formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, import residues for the formation of a cavity can be alanine (A), serine (S), threonine (T) and valine (V); and preferably the original residue for the formation of the cavity has a laige side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. For example, a T366W mutation in CH3 domain for the “knob’ ’ / protuberance chain, and a T366S / L368A / Y407V mutation in CH3 domain for tire “hole” / cavity chain. Additionally, tire KiH configuration may be coupled further mutations to pennit S-S disulfide linkage between tire two chains.

[0358] In some embodiments, the two or more arms (or chains) of immunoglobulin heavy chain constant regions (e.g., Fc polypeptides) can contain another symmetric-to-asymmetric steric complementarity design (e.g., HA- TF, ZW1), a charge-to-charge swap interaction (DD-KK), a chaige-to-steric complementarity swap plus additional long-range electrostatic interaction (e g., EW-RVT), or an isotype strand swap design (e.g., strand-exchange engineered domain (SEED)), or Xmab, 7.8.60. Electrostatic Steering, A107, or Duobody, so as to form heterodimeris / heteromultimers. Further description of these configurations and exemplary mutations / residues are seen in, for example, Front Immunol. 2016; 7: 394.

[0359] Further embodiments provide drat a multispccific antibody may further comprise one or more linkers, such as amino acid linkers. In some embodiments, at least one linker is between the Fc or the HSA and at least one of the antigen-binding sites. In some embodiments, at least one linker is positioned between two antigen-binding sites.

[0360] In some embodiments, a multispecific antibody comprises (1) a “knob-in-hole” IgG Fc domains with (2) a CD3-specific Fab on the N-terminus of the “knob” chain (or the “hole” chain) of the IgG Fc domain and (3) two ROR1 -specific VHH fused in tandem, optionally connected via a linker (VHH-linker-VHEI), on the N-terminus of the "hold” chain (or the "knob chain, respectively) of the IgG Fc domain. The two RORl-specific VHH fused in tandem herein may be two 2A11, two 5A1, or one 2A11 and one 5A1. two variants of 2A11, or two variants of 5A1.Exemplary multispecific antibodies of these embodiments include fusion proteins of FUSE-211, and FUSE-393, whose amino acid sequences are shown in Table 8. In some embodiments, the Fc domain is selected from Table 17A

[0361] In some embodiments, a multispccific antibody comprises (1) a “knob-in-holc” IgG Fc domains with (2) a CD3-specific Fab (comprising one constant and one variable domain of each of the CD3-specific heavy and the CD3-specific light chain) on the N-terminus of the ‘"knob” chain (or tire '’hole" chain) of the IgG Fc domain, (3) a first ROR1 -specific VHH fused to tire N-terminus of the “hole” chain (or the “knob” chain, respectively), and (4) a second RORl-specific VHH fused to the C-terminus of the CD3-specific light chain. The first and second ROR1- specific VHH can be identical or different, independently selected from 2A 11 , 5 A 1 , or a variant of2Al l or a variant of 5A1. Exemplary multispccific antibodies of these embodiments include fusion protein FUSE-394, whose ammo acid sequence is shown in Table 8. hr some embodiments, the Fc domain is selected from Table 17A.

[0362] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV PSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:532) is an exemplary’ heavy’ chain of an anti-CD3 Fab, and SEQ ID NO:479 is an exemplary light chain of an anti-CD3 Fab. Tin various embodiments, the anti-CD3 Fab is constant and sometimes present on the knob Fc and sometimes present on the hole Fc.

[0363] In some embodiments, a multispecific antibody comprises (1) a “knob-in-hole” IgG Fc domains with (2) a CD3-specific Fab (comprising one constant and one variable domain of each of the CD3-specific heavy’ and the CD3-specific light chain) fused to the N-terminal of the “knob” chain (or the “hole” chain) of the IgG Fc domain, (3) two RORl-specific VHH fused in tandem to the N-terminus of the “hole” chain (or the “knob” chain, respectively) of the IgG Fc domain, and (4) a third specific VHH fused to the C-terminus of the CD3-specific light chain. The two, fused-in-tandem RORl-specific VHH and the third RORl-specific VHH may be identical or different each independently selected from 2A 11 , 5 A 1 , or a variant of 2A 11 or a variant of 5 A 1. In other embodiments, the first and second RORl-specific VHH can be identical or different, independently selected from Table 14. hr some embodiments, the Fc domain is selected from Table 17A.

[0364] In some embodiments, a multispecific antibody comprises (1) a "knob-in-hole" IgG Fc domains with (2) a CD3-specific Fab fused to the N-terminus of the “knob” chain (or the “hole” chain) of the IgG Fc domains. (3) a first RORl-specific VHH fused to the N-terminus of the “hole” chain (or the “key” chain, respectively) of the IgG Fc domains, and (4) a second RORl-specific VHH fused to the N-terminus of tire CD3-specific light chain of the Fab. The first and second RORl-specific VHH can be identical or different, independently selected from 2A11, 5A1, or a variant of 2 A 11 or a variant of 5 A 1. In other embodiments, tire first and second R0R1 -specific VHH can be identical or different, independently selected from Table 14. In some embodiments, the Fc domain is selected from Table 17A.

[0365] In some embodiments, a multispecific antibody comprises (1) a “knob-in-hole” IgG Fc domains with (2) a CD3-specific Fab fused to the N-terminus of the “knob” chain (or the “hole” chain) of the IgG Fc domains,(3) a first R0R1 -specific scFV fused to the N-terminus of the “hole” chain (or the '‘knob” chain, respectively) of tire IgG Fc domains, and (4) a second RORl-specific scFV fused to the C-terminus of the “hole” (or the “knob” chain, respectively) of tire IgG Fc domains. The first and second RORl-specific scFv can be identical or different. In some embodiments, the Fc domain is selected from Table 17A.

[0366] In some embodiments, a multispecific antibody comprises (1) a “knob-in-hole” IgG Fc domains with (2) a CD3-specific Fab fused to the N-terminus of the “hole” chain (or the “knob” chain) of the IgG Fc domains, (3) a first RORl-specific VHH fused to the N-terminus of the “knob” chain (or the “hole” chain) of the IgG Fc domains, and (4) a second RORl-specific VHH fused to the C-terminus of the CD3-specific light chain of the Fab. The first and second ROR1 -specific VHH can be identical or different, independently selected from 2A11, 5A1, or a variant of 2A11 or a variant of 5A1. In other embodiments, the first and second RORl-specific VHH can be identical or different, independently selected from Table 147. In some embodiments, the Fc domain is selected from Table 17A.

[0367] In various embodiments, the disclosed multispecific antibodies (e.g., at least with immunospecificity towards ROR1 and CD3) can bind to R0R1 with an apparent binding affinity that generally increases (i.e., corresponding to a binding EC5o - a concentration of the antibody that results in 50% of maximum binding to a certain population of R0R1 -expressing target cells - that generally decreases) with an increase in the cell surface density of ROR1 of the target cells. That is, the multispecific antibodies can in various embodiments have a greater binding avidity towards ROR1 -expressing cells with a higher expression level of ROR1 than towards cells with a lower or zero expression level of ROR1.

[0368] In various embodiments, a system comprising the disclosed multispecific antibodies (e.g., at least with immunospecificity towards ROR1 and CD3) and a population of CD3-positive immune effector cells or a CD3- positive fraction of mononuclear cells or a population of mononuclear cells comprising a CD3-positive fraction can induce cytotoxicity against ROR1 -positive cells, i.e.. ROR1 -expressing tumor cells.

[0369] In some embodiments, the multispecific antibodies comprising an antigen-binding site that immunospecifically binds CD3 can bind CD3-epsilon on primary human T cells and / or primary cynomolgus T cells. In some embodiments, the multispecific antibodies comprising an antigen-binding site that immunospecifically binds CD3 activates cytotoxicity of primary human CD3+ T cells and / or primary cynomolgus CD3+ T cells, In general, CD8+ T cells are cytotoxic whereas CD4+ T cells primary secrete cytokines to “help” CD8+ T cells. In some instances, CD4+ T cells have also been reported to be cytotoxic (David Oh, et al.. Immunity, Volume 54, Issue 12, 14 December 2021, Pages 2701-2711). Cytotoxicity^ generally is associated with killing of target cells via the perforin / Granzyme pathway? that activates Caspase 3 and subsequent target cell apoptosis. Other mechanisms of target killing include but are not limited to activation of target cell express Fas, TNF-R and DR4. TCEs directly induce cytotoxicity and may also indirectly induce tire oilier said mechanisms of target killing (Sandra Ross et al., PLoS One. 2017 Aug 24;12(8):e0183390).

[0370] In some embodiments, the disclosed multispecific antibodies can induce cytotoxicity against ROR1- positive cells, i.e., R0R1 -expressing tumor cells, in the presence of a population of CD3-positive immune effector cells, a CD3-positivc fraction of mononuclear cells, or a population of mononuclear cells comprising a CD3-positivc fraction, In some embodiments, the disclosed multispecific antibodies can induce a greater cytotoxicity against R0R1 -positive cells that have a higher expression level of R0R1 than against R0R1 -negative cells or ROR1 -positive cells that have a lower expression level of ROR1, when in the presence of a population of CD3-positive immune effector cells, a CD3- positive fraction of mononuclear cells, or a population of mononuclear cells comprising a CD3-positive fraction.

[0371] Further embodiments provide that in place of, or in addition to, having CD3-specificity. a multispecific antibody of tire invention may have a specificity against another activation receptor and / or costimulatory receptor and / or co-activation receptor typically expressed on an immune cell, besides having a specificity against R0R1. For example, an activation receptor can be expressed on a T cell, such that the multispecific antibody may be a T cell engager that immunospecifically binds to the activation receptor on the T cell and activates the cytotoxicity or a cytokine expression property of the T cell. Alternatively, an activation receptor can be expressed on a natural killer (NK) cell, such that the multispecific antibody may be a NK cell engager that immunospecifically binds to the activation receptor on the NK cell and activates tire cytotoxicity of the NK cell. Additionally, an activation receptor may be expressed on multiple immune cells such as T cells, NK cells and Dendritic cells (DC). Hence, the multispecific antibodies may be trifiinctional immune cell engagers, or trispecific antibodies, which bind to two or more activation receptors (such as two or more of CD3, CD16, y9 TCR, 82 TCR or 81 TCR, NKp46, CD137 and CD40), thereby inducing activity of the immune cells, and also bind to R0R1 -positive cells, thereby directing the immune cell activity towards tire ROR-positive cells.

[0372] Exemplary activation receptors that can be immunospecifically bound by an antigen-binding site of the multispecific antibodies disclosed herein include, but are not limited to CD 16, y9 TCR, 82 TCR or 81 TCR, NKp46, CD137, CD40 or NKG2D. Exemplary' antigen-binding fragments against said activation and / or costimulatory and / or co-activation receptors include scFv, VH, VL, VHH, Fab, etc., described in various publications including U.S. Pat. No. 9,035,026 and Gauthier et al., 2019, Cell 177. 1701-1713. Additional exemplary antigen-binding antibodies and / or fragments against said activation and / or costimulatory and / or co-activation receptors include those noted in Table 18. Of those listed as the antigen-binding antibodies, their VHH or Fab regions can be used as the antigen-binding site of the multispecific antibodies disclosed herein.

[0373] Additional embodiments provide R0R1 -specific antibody-drug conjugates (ADCs), which comprises an anti-RORl antibody, or an R0R1 -binding fragment thereof, disclosed herein, and a drug unit that is conjugated or bound to the anti-RORl antibody or R0R1 -binding fragment thereof. Optionally, a linker unit is positioned between the anti-RORl antibody or R0R1 -binding fragment thereof and the drug unit, and the linker unit may be an amino acid linker or a chemical moiety linker. Exemplary? drug units can be cytotoxic agents such as antitumor drugs or chemotherapy agents, including small molecules as well as siRNA.

[0374] In additional embodiments, the multispecific antibody, multispecific antigen-binding fragment, or composition comprising tire same may also contain a further therapeutic agent / therapy, as necessary for the particular disorder being treated. Preferably, the multispccific antibody or multispccific antigen-binding fragment thereof and tire further therapeutic agent have complementary activities that do not adversely affect each other. In some embodiments, the further therapeutic agent / therapy is a chemotherapeutic agent or a radiation therapy. Combined administration of the disclosed multispecific antibodies or multispecific antigen-binding fragments thereof and the other therapeutic agent may be simultaneous, separate or sequential, in any order. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0375] The dose of the multispecific antibody, multispecific antigen-binding fragment, or composition comprising the same depends on the desired effect, the duration of the treatment, and the route of administration used. For example, a therapeutically effective amount of a multispecific antibody disclosed herein includes one or more doses, wherein a dose is in the range of about 10-50 mg, 50-100 mg, 100-150 mg, 150-200 mg, 100-200 mg, 200-300 mg, 300-400 mg, 400-500 mg, 500-600 mg, 600-700 mg, 700-800 mg, 800-900 mg, 900-1000 mg, 1000-1100 mg, 1100-1200 mg, 1200-1300 mg, 1300-1400 mg, 1400-1500 mg, 1500-1600 mg, 1600-1700 mg, 1700-1800 mg, 1800- 1900 mg, 1900-2000 mg, 2000-2100 mg, 2100-2200 mg, 2200-2300 mg, 2300-2400 mg, 2400-2500 mg, 2500-2600 mg, 2600-2700 mg, 2700-2800 mg, 2800-2900 mg or 2900-3000 mg. In another embodiment, a therapeutically effective amount of a multispecific antibody disclosed herein includes one or more doses, wherein a dose is in the range of 0.001-0.005 mg / kg, 0.005-0.01 mg / kg, 0.01-0.02 mg / kg, 0.02-0.04 mg / kg, 0.04-0.06 mg / kg, 0.06-0.08 mg / kg, 0.08-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, 15-20 mg / kg, 20-25 mg / kg, 25-30 mg / kg, 30-35 mg / kg, 35-40 mg / kg, 40-45 mg / kg, 45-50 mg / kg, 10-50 mg / kg, 50-100 mg / kg, 100-150 mg / kg, 150-200 mg / kg, 100-200 mg / kg, 200-300 mg / kg. 300-400 mg / kg, 400-500 mg / kg, 500-600 mg / kg, 600-700 mg / kg, 700-800 mg / kg, 800-900 mg / kg, 900-1000 mg / kg. 1000-1100 mg / kg, 1100-1200 mg / kg, 1200-1300 mg / kg, 1300-1400 mg / kg, 1400-1500 mg / kg, 1500-1600 mg / kg, 1600-1700 mg / kg, 1700-1800 mg / kg, 1800-1900 mg / kg, 1900-2000 mg / kg, 2000-2100 mg / kg, 2100-2200 mg / kg, 2200-2300 mg / kg, 2300-2400 mg / kg, 2400-2500 mg / kg, 2500-2600 mg / kg, 2600-2700 mg / kg, 2700-2800 mg / kg, 2800-2900 mg / kg or 2900-3000 mg / kg. In some embodiments, a therapeutically effective amount includes two, three, or more doses administered on a daily, weekly, biweekly, monthly, quarterly, or yearly frequency; or continued when evaluation of tire severity of the disease shows improvement, compared to before the last dose, until tire disease is successfiilly treated or the subject shows no symptoms or signs of the disease.

[0376] Table 8. Amino acid sequences of exemplary fusion proteins. Polypeptide 1 comprises the ‘hole’ of the KiH Fc; Polypeptide 2 comprises the ‘knob’ of tire KiH Fc; and Polypeptide 3 comprises the light chain. The VHH may be included in Polypeptide 1, polypeptide 2 and / or polypeptide 3 with a linker between them, hr this table the anti- CD3 domain is on the N-temiinal of tire knob Fc or hole Fc.Table 9. Polynucleotide sequences encoding the fusion proteins corresponding to those in Table 8.Table 10. Amino acid sequences of exemplary VHH.Table 1 1 . Polynucleotide sequences encoding the VHH shown in Table 10.Table 12. Yield of expressions as demonstrated in Examples for some fusion proteins.Table 13. Melting temperature (Tm) of exemplary fusion proteins.Table 14. Additional exemplary VHH sequences used in in accordance with various embodiments of the invention. All sequences are humanized sequences except for SEQ ID NO:7 and SEQ ID NO:8.Table 15A CDRsTable 17A. Polypeptide 1 comprises an Fc domain; Polypeptide 2 comprises an Fc domain; and Polypeptide 3 comprises the light chain. The VHH antibody may be included in Polypeptide 1 , polypeptide 2 and / or polypeptide 3, and can utilize a linker between them. In this table the anti-CD3 domain is on the N-terminal of tire Fc. The “(VHH antibody)” can be selected from Table 14.Table 17B. Nucleotide sequences encoding the polypeptides in Table 17A.Table 18.IL-15 variants and IL-15 fusion proteins

[0377] Various embodiments of the present invention provide for IL- 15 variants.

[0378] Various embodiments provide for an interleukin 15 (IL-15) variant comprising the sequence ofFormula I:thereof. In various embodiments, any combination thereof is any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 combinations thereof.

[0379] In various embodiments of the IL-15 variant, X49is R or K, and X53is G, K, I or A; (SEQ ID NO:564).

[0380] In various embodiments of the IL-15 variant, X30is N (SEQ ID NO:565).

[0381] In various embodiments of the IL- 15 variant, XMis Q (SEQ ID NO: 566).

[0382] In various embodiments of the IL-15 variant, Xi is G (SEQ ID NO:567).

[0383] In various embodiments, tire IL- 15 variant is a variant selected from Table 1A or Table 3 A.

[0384] In various embodiments, the IL- 15 variants are not IL- 15 variants described in U.S. Patent Application Publ. No. US 2019 / 0263877, the variants which are herein incorporated by reference.

[0385] Also provided herein are fusion proteins comprising IL- 15 variants and a scaffold polypeptide.Various embodiments of the invention provide for a fusion protein, comprising an IL- 15 variant as described herein: and a scaffold polypeptide. In various embodiments, the fusion protein comprises two or more IL- 15 variants as described herein; and a scaffold polypeptide. In various embodiments, the two or more IL-15 variants can be 3, 4, 5 or 6 IL-15 variants. In various embodiments, the two or more IL-15 variants can be up to 8 IL-15 variants.

[0386] In various embodiments, the fusion protein further comprises one or more immune checkpointtargeting fragment.

[0387] In various embodiments, the immune checkpoints include but are not limited to PD- 1 , PD-L 1, CTLA-4, LAG-3. In various embodiments, one or more immune checkpoint-targeting fragment comprises immune checkpoint-targeting fragments of known antibodies. Examples of anti-PDl fragments include fragments (E.g. Fab, Fv) from pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalirnab (JS001). lipustobart (LZM009). retifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tebotelimab (MGD013), cadonilimab (AK104). vudalimab (XmAb20717), tislelizumab, or PF-06801591. Additional examples of anti-PDl fragments also include fragments (e g., Fab, Fv) from vopratelimab, camrelizumab, sintilimab, AMP-224, AMP-514, and Acrixolimab. Examples of anti-PD- L1 antibodies include but are not limited to garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolirnab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab,BMS-936559, CK-30L and M7824. Thus, for example, the fusion protein comprises an anti-PDl fragment from an anti-PDl antibody (e.g., Fab, Fv), and an IL-15 variant, and optionally, a linker.

[0388] In various embodiments, the IL- 15 variant is fused to tire C-tcrminus of the scaffold polypeptide. In other embodiments, the IL- 15 variant is fused to the N-terminus of the scaffold polypeptide .

[0389] In still other embodiments, wherein the scaffold polypeptide is an antibody, the IL- 15 -variant is fused to any one of the C-terminus or N-terminus of the heavy chain or light chain, or fused to any one of the CH2 domain, or hinge region (e.g., between tire Cn2 and Cnl domains).

[0390] hr various embodiments, the scaffold polypeptide is an antibody or a fragment thereof, In various embodiments, the antibody is an IgA. IgM, IgG. or IgE antibody.

[0391] In various embodiments, the antibody is an anti-PD-1 antibody or anti-PD-Ll antibody. Examples of anti-PD-1 antibodies include but are not limited to pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD-100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), rctifanlimab (MGA012), Sym021, dostarlimab (TSR-042), tcbotclimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, PF-06801591, and anti-PDl antibody expressing pluripotent killer T lymphocytes (PIK-PD-1). Examples of anti-PD-Ll antibodies include but are not limited to garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX-1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824.

[0392] In various embodiments, the scaffold polypeptide is a Fc region or a fragment thereof. In various embodiments, the scaffold polypeptide is a Fc region or a fragment thereof and the scaffold polypeptide does not comprise an Fab.

[0393] In various embodiments, two arms (or chains) of immunoglobulin heavy chain constant regions (e.g.,Fc polypeptides) can be heterodimerized by creating “knobs-in-holes” (KiH) mutations in the CH3 domain. This structural feature in the polypeptide amis allows for assembly of two half antibodies (e.g., Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within the CH3 domain, and tire one polypeptide contains an engineered protuberance ("knob”) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and another polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue. In some embodiments, the engineered interface of a heteromultimer includes at least two protuberance-into-cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972 and C. Chothia. J. Mol. Biol. 105: 1-14. 1975. For example, import residues for the formation of a protuberance can be arginine (R), phenylalanine (F). ty rosine (Y) and tryptophan (W); and preferably the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. As another example, importresidues for the formation of a cavity can be alanine (A), serine (S), threonine (T) and valine (V); and preferably the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. For example, a T366W mutation in CH3 domain for the “knob’ / protuberance chain, and a T366S / L368A / Y407V mutation in CH3 domain for tire “hole’ / cavity chain. Additionally, tire KiH configuration may be coupled further mutations to pemrit S-S disulfide linkage between the two chains, hr various aspects wherein tire protein / polypeptide is a heterodimer of a the KiH configuration, the IL-18 (or its fragment variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) immunoglobulin heavy chain constant regions / chains (i.e. knob or hole).

[0394] As such, in some embodiments, the Fc region is aknobs-in-holc (KiH) Fc.

[0395] In various embodiments, the IL- 15 variant is fused to the knob of the KiH Fc. In various embodiments, tire IL-15 variant is fused to tire hole of the KiH Fc.

[0396] In various embodiments, the Fc region is an Fc region from an IgG4, knobs-in-hole (KiH) Fc, orIgGl . In various embodiments, Fc region can be a human IgGl , IgG2, or IgG4.

[0397] In some embodiments, the two or more arms (or chains) of immunoglobulin heavy chain constant regions (e.g., Fc polypeptides) can contain another synmretric-to-asymmetric steric complementarity design (e.g., HA- TF, ZW1), a charge-to-charge swap interaction (DD-KK), a charge-to-steric complementarity swap plus additional long-range electrostatic interaction (e.g., EW-RVT), or an isotype strand swap design (e.g., strand-exchange engineered domain (SEED)), or Xmab, 7.8.60, Electrostatic Steering, A107, or Duobody, so as to form heterodimeris / heteromultimers. Further description of these configurations and exemplary mutations / residues are seen in Front Immunol. 2016; 7: 394.

[0398] In various embodiments, the scaffold polypeptide of the fusion protein comprises a globular protein, human semm albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, or a fragment thereof.

[0399] In various embodiments, one or more polypeptides can be inserted between the IL- 15 variant and tire antibody of the IL-15 fusion proteins. In various embodiments, the polypeptide can be inserted or conjugated at the N- terminus, at the C-terminus, or both the N-terminus and C- terminus of the antibody. In various embodiments, the polypeptide comprises a polypeptide linker conjugating the IL- 15 variant and tire antibody.

[0400] hr various embodiments, one or more polypeptides can be inserted between the IL- 15 variant and theFc region of the IL-15 fusion proteins. In various embodiments, the polypeptide can be inserted or conjugated at the N- tenninus. at the C-tenninus. or both the N-terminus and C- terminus of the Fc region. In various embodiments, the polypeptide comprises a polypeptide linker conjugating the IL- 5 variant and the Fc region. In these embodiments, tire fusion protein does not include Fab region of an antibody.

[0401] The antibodies that can be fused to IL- 15 can include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, and single chain (ScFv). The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibodies are human or humanized antibodies.

[0402] In various embodiments, the IL-15 fusion proteins are IL-15 fusion proteins listed in Table 1A. That is, an IL- 15 fusion protein comprising polypeptide 1, polypeptide 2, and polypeptide 3.

[0403] In various embodiments, the IL- 15 fusion protein comprises an IL- 15 variant listed in Table 1A orTable 3A.

[0404] In various embodiments, tire IL- 15 fusion protein comprises polypeptide 1, polypeptide 2, and polypeptide 3 listed in Table 1 A, except the linker in polypeptide 2 is a different peptide linker. For example, the linker can be a flexible linker that is generally about 10 to 25 amino acids in length. Additional examples of linkers include but are not limited to a dimer of two amino acids, a tri-mer of three amino acids, or a peptide selected from tire group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX, (SEQ ID NO:236))n, and (X,GGGG (SEQ ID NO:317))n wherein X,. is Q, A, E or S and n=l-5 or an integer larger than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))n where n is an integer between 1 and 5. thereby an amino acid linker of 25 amino acids or shorter in length.

[0405] Provided herein are methods of producing the IL- 15 variants and IL- 15 fusion proteins.

[0406] Various embodiments provide for a polynucleotide encoding an IL-15 variant of the present invention described herein. For example, the nucleic acid sequences may encode in a 5’ to 3’ direction, the IL- 15 variant or the IL- 15 fusion protein.

[0407] Various embodiments provide for a polynucleotide encoding an IL- 15 fusion protein of the present invention described herein. For example, the nucleic acid sequences may encode in a 5’ to 3’ direction, tire IL- 15 variant.

[0408] As such, the polynucleotide encoding an IL- 15 fusion protein of the present invention comprises polynucleotide 1, polynucleotide 2 and polynucleotide 3 listed in Table 2A. In other embodiments, the polynucleotide encoding an IL- 15 fusion protein of the present invention comprises polynucleotide 1, polynucleotide 2 and polynucleotide 3 listed in Table 2A, except the nucleotides encoding the linker encodes a different linker.

[0409] For example, the linker can be a flexible linker that is generally about 10 to 25 amino acids in length. Additional examples of linkers include but are not limited to a dimer of two amino acids, a tri-mer of three amino acids, or a peptide selected from tire group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX, (SEQ ID NO:236))n, and (X GGGG (SEQ ID NO:317))n wherein Xλ is Q, A, E or S and n=l-5 or an integer huger than 5. hr some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237))nwhere n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in length. As such, tire nucleotides encoding the linker in polynucleotide 2 will encode one of these aforementioned linkers instead.

[0410] Exemplary polynucleotides arc in Tabic 2A.

[0411] Various embodiments also provide for an expression vector comprising any one of tire polynucleotides described herein. Thus, the polypeptide encoding the IL-15 variant or IL-15 fusion proteins may also be integrated within a replicable expression vector. Hence, a vector encoding the fusion protein is also provided, which may express the fusion protein in, for example, a bacterial host, an intended recipient, or both.

[0412] Additional embodiments provide cells transformed or transfected with one or more nucleic acid molecules encoding tire IL-15 variant or IL-15 fusion protein. The cell can be a prokaryotic cell. Or the cell is a eukaryotic cell, preferably a mammalian cell, and more preferably a human cell.

[0413] In various embodiments, the cell is a mammalian cell. Mammalian cells that are usefi.il include but are not limited to Chinese Hamster Ovary (CHO) cells or HEK-293 cells.

[0414] In various embodiments, tire cell is a bacterial cell or a yeast cell.

[0415] Various embodiments provide for a method of producing an IL- 15 variant, comprising: culturing any one of tire cells described herein, in cell culture medium to allow the 11-15 variant to be produced, and optionally secreted into the cell culture medium.

[0416] In various embodiments, the method further comprises isolating the IL- 15 variant from the cell or from the cell culture medium. In various embodiments, the method further comprises purifying the IL-15 variant.

[0417] Various embodiments provide for a method of producing an IL-15 fusion protein, comprising: culturing any one of the cells described herein, in cell culture medium to allow the 11-15 fusion protein to be produced, and optionally secreted into the cell culture medium.

[0418] In various embodiments, the method further comprises isolating the IL- 15 fusion protein from the cell or from the cell culture medium. In various embodiments, the method further comprises purifying the IL-15 fusion protein.

[0419] In some embodiments, using a Chinese hamster ovary (CHO) expression system, the IL- 15 variant or fusion protein is produced via a process including the steps of: (1) cell recovery, which may be to recover frozen CHO cells via water bath at 37°C; (2) cell subculturing, which may be to sub-culture the cells and adjust the cell density to 6x l06 / ml for transfection; (3) transfection and expression, using a solution 1 (in which a plasmid is diluted with a diluting agent), a solution 2 (in which a transection reagent is diluted with a / the diluting agent), and tiien mixing the solution 1, the solution 2 and the CHO cells, followed by incubating the mixture at a shaker for expression for 12-14 days at 32°C to collect the supernatant of the culture after centrifuge.

[0420] Tn some embodiments, a purification process is performed after the expression of the fusion protein.In some embodiments, a purification process includes the steps of: (1) washing a column with a binding buffer (10 times volume) at a flow rate of 1 Ml / min; (2) loading a fusion-protcin-containing sample into tire column at a flow rate of 1 Ml / min; (3) washing the column with 10x volumes of PBS buffer with a flow rate of 1 Ml / min; (4) eluting the protein from the column with 40 Mm sodium citrate (Ph3.4); optionally the elution sample may be collected into tubes (Iml / min) and measured for optical density (OD) using NanoDrop at 280 run; and (5) performing dialysis, e.g., against PBS buffer in a dialysis bag overnight.

[0421] Various embodiments of tire invention provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering an IL- 15 variant of tire present invention as described herein, to a subject in need thereof.

[0422] Various embodiments of the invention provide for a method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering an IL-15 fusion protein of the present invention as described herein, to a subject in need thereof.

[0423] In various embodiments, tire subject has cancer.

[0424] Various embodiments of the invention provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering an IL-15 variant of the invention as described herein to a subject in need thereof.

[0425] Various embodiments of the invention provide for a method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering an IL-15 fusion protein of tire invention as described herein to a subject in need thereof.

[0426] In various embodiments, the disease or condition is cancer.Pharmaceutical compositions

[0427] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the fusion protein. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic. and desirable, and includes excipients that arc acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in tire case of an aerosol composition, gaseous.

[0428] In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmacally acceptable salts with pharmaceutically acceptable bases. Tire tenn “pharmacally acceptable salts, esters, amides, and prodrugs” as used herein refers to Arose carboxylate salts, amino acid addition salts, esters, amides, and prodrags of the compounds of the present inventionwhich are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use of tire compounds of the invention. The term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base fonn with a suitable oiganic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Beige S. M., et al. (1977) J. Phann. Sci. 66, 1, which is incorporated herein by reference).

[0429] The term “pharmaceutically acceptable esters” refers to the relatively nontoxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The tenn is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.

[0430] As used herein, “pharmaceutically acceptable salts or prodrugs” are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0431] The term “prodmg” refers to compounds that are rapidly transformed in vivo to yield the functionally active one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof. A thorough discussion is provided in T. Higachi and V. Stella, “Pro-drags as Novel Delivery Systems,” Vol. 14 of the A. C. S. Symposium Series, and in Bioreversible Carriere in: Drag Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. A prodrag of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be designed to alter the metabolic stability or the transport characteristics of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to mask side effects or toxicity, to improve the flavor of a compound or to alter other characteristics or properties of a compound. By virtue of knowledge of phannacodynamic processes and drug metabolism in vivo, once a pharmacally active fonn of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, those of skill in tire pharmaceutical art generally can design prodrags of the compound (see. e.g., Nogrady (1985) Medicinal Chemistry A BiochemicalApproach, Oxford University Press, N. Y., pages 388-392). Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs,’’ ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl and glycerol esters of the corresponding acid.

[0432] In various embodiments, tire pharmacal compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.

[0433] 'Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.

[0434] “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular. intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0435] Via the enteral route, the pharmacal compositions can be in the fomi of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.

[0436] Via tire topical route, tire pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the fomi of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication.

[0437] Viatlre ocular route, they may be in the fomi of eye drops.

[0438] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in earn ing or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in tiiat it must be compatible widi the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, alleigic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0439] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or symp for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0440] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule fonns. When a liquid carrier is used, the preparation will be in tlie fomi of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0441] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in tenns of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to tire characteristics of the therapeutic compound (including activity, phannacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age. sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in tire formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting tire dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20thedition, Williams & Wilkins PA. USA) (2000).Kits

[0442] The present invention is also directed to a kit to treat various diseases. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including fusion proteins, as described above.

[0443] The exact nature of the components configured in the inventive kit depends on its intended purpose.For example, some embodiments are configured for the purposes of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells. Some embodiments arc configured for the purpose of treating cancer. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, fann animals, domestic animals, and laboratory animals.

[0444] Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to activate, promote differentiation or promote expansion of T cells, B cells or natural killer (NK) cells, to treat diseases, including cancer. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0445] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical stinctures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing IL- 15 variants or fusion proteins. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.EXAMPLES

[0446] The following examples are provided to better illustrate tire claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.EXAMPLESIL15 / IL18 VARIANT EXAMPLESExample 1

[0447] As shown in figure 2, we assessed the impact of an exemplary anti-PDl-pro-IL-18 antibody, one of three exemplary anti-PDl-mutant-IL-15 antibodies or the combination of the anti-PDl-pro-IL-18 antibody and of each of the three mutant IL- 15 antibody fusions on IFNy release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (Pelicluster CD3, which is capable of crosslinking CD3 and activating T cells independent of FcyR binding) for 72 hours. For anti-PDl-pro-IL-18, the exemplary test article was Fuse694. which incorporates one pro-IL-18 (granzyme B cleavable IL18mutAS) on the C-terminal knob or hole of an IgGl-LALA version of nivolumab (Fuse691). For anti-PDl-mutant-IL-15, the three test articles were Fuse765 (FIG. 2A, open triangle, dashed line), Fuse773 (FIG. 2B, open triangle, dashed line) and Fuse774 (FIG. 2C, open triangle, dashed line), which respectivelyincorporated one IL-15m4, IL15m9 or IL 15m 10 on the C-terminal knob or hole of Fuse691. FIGs 2A-2C are nonlinear x-y plots of test article concentration versus IFNy release.

[0448] In each plot, (1) tire Hen Egg Lysosyme (HEL) specific antibody, anti-HEL (gray circle), was used as the human IgGl isotype control and was associated with background IFNy release. Fuse691 (open square, dashed line) was observed to induce mild IFNy release in this system, equating to a maximum value of about 1200 pg / ml. Targeting pro-IL-18 or any of tire three IL-15 mutants alone to PD1 using Fuse694, Fuse765, Fuse773 and Fuse774 resulted in only a modest increase in maximum observed IFNy release relative to Fuse691 with values of approximately 1900 pg / ml, 3000 pg / ml, 2800 pg / ml and 1700 pg / ml, respectively, hr contrast, the combination of Fuse765 plus Fuse694 (black triangle) , Fuse773 plus Fuse694 (black triangle) and Fuse774 plus Fuse694 (black triangle) resulted in maximum observed IFNy release values of approximately 12,000 pg / ml, 11,500 pg / ml and 7,400 pg / ml, respectively. These values correspond to 4-6-fold that of anti-PDl-pro-IL-18 alone (Fuse694) and or any of the anti-PD 1-mutant- IL15 variants (Fuse765, Fuse773 and Fuse774) indicating that the combined impact of targeting both IL-15 and IL-18 to PD1 in this system is synergistic. Interestingly, the EC50- IFNy release, although only semi-quantitative for Fuse691 and the single cytokine fusions to anti-PD 1 , appeared to cluster within 2-3-fold of each other.

[0449] A summary tabic of EC50, Emax and AUC (area under the curve) values is show n in FIG. 2D.Example 2

[0450] As shown in figure 3, we assessed tire impact of an exemplary anti-PDl-pro-IL-18 antibody, an exemplary anti-PD 1-mutant-IL- 15 antibody and an anti-PD 1 antibody incorporating both tire aforementioned exemplary pro-IL-18 and mutant IL- 15, on IFNy release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (PeliCluster CD3, which is capable of crosslinking CD3 and activating T cells independent of FcyR binding) for 72 hours. For anti-PDl-pro-IL-18, the exemplary test article was Fuse694 (black square). For anti-PD 1 -mutant-IL- 15, the exemplary test article was Fuse696 (black diamond), which incorporated one IL-15m2 on the C-terminal knob or hole of the anti-PDl human IgGl LALA antibody, Fuse691 (open square, dashed line). Finally, Fuse697 (black triangle), incorporated one pro-IL18 on tire C-terminal knob and one IL-15m2 on the C-terminal hole of Fuse691. FIG 3 is non-linear x-y plot of test article concentration versus IFNy release. The human IgGl antibody, anti-HEL (gray circle), was used as the isotype control and was associated with background IFNy release. Fuse691 (open square, dashed line) was observed to induce mild IFNy release in this system, equating to a maximum value of about 830 pg / ml. Targeting pro-IL-18 alone to PD 1 using Fuse694 resulted in only a modest increase in maximum observed IFNy release relative to Fuse691 with a value of approximately 2,000 pg / ml. Targeting IL-15m2 alone to PD1 using Fuse696 resulted in a more pronounced increase in maximum observed IFNy release relative to Fuse691 with a value of about 9,000 pg / ml. Fuse697 induced a maximum observed IFNy release values of approximately 21,700 pg / ml, which is about 11 -fold greater than that observed for Fuse694 and 2.5-fold greater than that observed for Fuse696. Importantly, maximum observed IFNy release for Fuse697 equated to more than the sum of the maximum IFNy release valuesassociated with Fuse694 plus Fuse696 indicating that IL- 15 and IL- 18 acted in a synergistic fashion when coincorporated into anti-PDl. Interestingly, unlike the EC50-IFNy release for the combination of anti-PDl-pro-IL-18 and anti-PDl-mutant-IL15 relative to Fuse691 and the single cytokine fusions to anti-PDl, tire EC50- IFNy release for Fuse697 was obviously left shifted (more potent) by at least 30-fold relative to Fuse691, Fuse694 or Fuse696. A summary table of EC50, Emax and AUC (area under the curve) values is shown below the x-y plot.Example 3

[0451] As shown in figure 4, we assessed the capacity of an NKG2D x R0R1 bispecific antibody (bsAb) with or without the incorporation of an exemplary pro-IL18 and IL 15 mutant to induce NK cell mediated tumor cell killing and IFNy release. The NKG2D x R0R1 knobs into holes bsAb was human IgGl LALA (parent bsAb without cytokines incorporated was Fuse926) incorporated pro-IL18 (granzyme B cleavable IL18mutAV) on the N-terminal hole and IL15m9-l on the C-tenninal hole (Fuse916). Enriched NK cells were expanded from human PBMC (normal donor) and used as the effector cells. The ROR1+ tumor cells were MDA-MB-231 stably expressing firefly luciferase and Egfp (MDA-GL). NK cells were mixed with MDA-GL at an effector to target ratio (E:T) of 5: 1 for 48 hours. Tumor cell killing was assessed via luciferase activity and IFNy release was measured in the culture supernatants via ELISA. FIGs 4A and 4B are non-linear x-y plots of tumor cell killing versus test article concentration and IFNy release versus test article concentration, respectively. Relative to Fuse926 (black square), Fuse916 (black triangle) induced -50% more maximum killing and ~7-fold more maximum IFNy release. Tire potency (defined as the EC50-killing or EC50- IFNy release) of Fuse916 was also enhanced relative to Fuse916 in the orders of 5-fold and >10-fold for tumor cell killing and IFNy release, respectively. The anti-HEL Ab serves as a negative isotypc control and Fuse923 (black diamond), a 982-TCR x R0R1 bsAb incorporating pro-IL18 and IL15m9-l in tire same orientation was used as the non-targeted cytokine incorporating test article. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.Example 4

[0452] As shown in figure 5. we assessed the impact of targeting an exemplary IL-15 variant and an exemplary pro-lL18 to NKG2D on NK cell expansion in PBMC. Both NK cell frequency and numbers were measured after 12 days of incubating PBMC from a healthy human donor with the test articles described below. NK cells were defined as CD56+ / CD3- cells. The antibody fusion proteins tested were Fuse 926 (black diamond), which consists of an NKG2D x R0R1 bsAb and Fuse916 (black triangle), which consists of the same bsAb but also incorporates IL-15m9-l and pro-IL18 (granzyme B cleavable IL18mutAV). Fuse923 (black square), a cytokine bsAb fusion similar to Fuse923 that targets the 52 region of the y982 TCR was used as the non targeted control and anti-HEL (gray circle) was used as the isotype matched negative control. Of note, all bsAbs contained the LALA mutation and therefore did not bind nor activate CD 16 expressed on NK cells. As such, the expansion of NK cells was limited to targeting of the cytokines toNKG2D only without co-ligation of NKG2D and CD16, reported to enhance one another's activity www.sciencedirect.corn / science / article / pii / S0006497120678016. FIG. 5A and FIG. 5B are x-y plots of NK cell percentages and NK cell numbers measured on day 12, respectively, illustrated as a function of a titration of each test article. Relative to tire Fuse926, 100 Nm of Fuse916 induced about a 6-fold expansion of NK cells.Example 5

[0453] As shown in figure 6, we assessed the impact of targeting an exemplary IL- 15 variant and an exemplary pro-IL18 to NKp46 on NK cell expansion in PBMC. Both NK cell frequency and numbers were measured after 12 days of incubating PBMC from a healthy human donor with the test articles described below. NK cells were defined as CD56+ / CD3- cells. Tire antibody fusion proteins tested were Fuse 943 (black triangle), which consists of a monovalent NKp46 Ab which incorporates IL-15m9 and pro-IL18 (granzyme B cleavable IL18mutAV). Fuse944 (black square), was used as tire non taigeted control in which the NKp46 binder was omitted from tire construct. Fuse320 (black diamond) , an NKp46 x EGFR bsAb incorporating the same NKp46 binder but no cytokines served as the targeted / no cytokine control (note that we did not detect expression of EGFR in PBMC; data not shown) and anti- HEL (gray circle) was used as tire isotype matched negative control. Of note, all bsAbs contained the LALA mutation and therefore did not bind nor activate CD16 expressed on NK cells. As such, the expansion of NK cells was limited to taigeting of the cytokines to NKp46 only without co-ligation of NKp46 and CD 16. reported to enhance one another's activity' www. scicnccdircct.com / scicncc / articlc / pii / S0006497120678016. FIG. 6A and FIG. 6B are x-y plots of NK cell percentages and NK cell numbers measured on day 12, respectively, illustrated as a function of a titration of each test article. Relative to tire Fuse320, 100 Nm of Fuse943 induced about a 5-fold expansion of NK cells.Example 6

[0454] As shown in figure 7, we assessed the capacity of an exemplary IL- 15 variant and / or an exemplary pro-IL18 targeted to ',.'932 T cells via a Vy9V82 TCR x ROR1 bsAb cytokine fusion to induce expansion of 932 T cells in human PBMC.

[0455] Both y982 cell frequency and numbers were measured after 12 days of incubating PBMC from a healthy human donor with the test articles described below. Given that ROR1 is not expressed on any cells in PBMC (note that ROR 1 is expressed on pre-B cells in human bone marrow), expansion observed was considered independent of Vy9V62 TCR crosslinking via ROR1.

[0456] The antibody fusion proteins tested were cytokine fusions to a Vy9V82 TCR x R0R1 bsAb (Fuse966; black diamond) incorporating (1) 1L-I5m9-1 (Fuse968; black square), (2) pro-lL18 (granzyme B cleavable IL18mutAV) (Fuse967; black reverse triangle) and (3) IL-15m9-l and pro-IL18. Fuse944 (open triangle, dashed line), was used as the non taigeted control in which tire VY9V82 TCR x R0R1 cassette was omitted from the construct. An anti-HEL (gray circle) was used as tire isotype matched negative control.

[0457] FIG. 7 A and FIG. 7B are x-y plots of 7982 T cell percentages and y982 T cell numbers measured on day 12, respectively, illustrated as a function of a titration of each test article. Relative to the Fuse966, 100 Nm of Fuse923 induced about a 12-fold expansion of y982 T cells.Example 7

[0458] As shown in figure 8, we assessed the capacity of an exemplary IL- 15 variant and / or an exemplary pro-IL18 targeted to y982 T cells via a Vy9V32 TCR x R0R1 bsAb cytokine fusion to induce y982 T cell mediated IFNy release and tumor cell killing. The assay design was as such: T962 T cells enriched / expanded from the PBMC of a healthy human donor was mixed with the R0R1+ tumor cell, MDA-MB-231 stably expressing Egfp and firefly lucerifase at an E:T ratio of 5: 1. After 48 hours, tumor cell cytotoxicity was assessed via luciferase activity (i.e. tumor cell viability) and IFNy release was measured in the supernatant using a standard ELISA protocol. The antibody fusion proteins tested were cytokine fusions to a Vy9V82 TCR x R0R1 bsAb (Fuse966; open reverse triangle, dashed line) incorporating (1) IL-15m9-l (Fuse968 ; open triangle, dashed line), (2) pro-IL18 (granzymc B cleavable IL18mutAV) (Fuse967 ; black reverse triangle) and (3) IL-15m9-l and pro-IL18. Fuse944 (black diamond), was used as the non targeted control in which the Vy9V82 TCRx R0R1 cassette was omitted from tire construct. An anti-HEL (gray circle) was used as tire isotype matched negative control. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 8A) or IFNy release (FIG. 8B). Relative to Fuse966. the incorporation of cytokine(s) into the bsAb enhanced both tumor cell killing and IFNy release in the rank order of Fusc923>Fusc967>Fusc968>Fusc966 indicating that the combination of IL-18 and IL-15 was the most potent. The differential readout between test articles was more apparent for IFNy release compared to tumor cell killing. This is likely due to the high E:T ratio of 5: 1 at which maximum tumor cell killing may be reached at a lower y982 T cell signaling threshold than IFNy release. Summan tables of EC50, Emax and AUC (area trader the curve) values are shown below each x-y plot.Example 8

[0459] As shown in figure 9, we assessed the impact of targeting an exemplary IL- 15 variant and an exemplary IL18 variant to NKp30 plus or minus NKp46 on NK cell expansion from PBMC. Both NK cell frequency and numbers were measured after 21 days of incubating PBMC from a healthy human donor with 100 Pm of the test articles described below. NK cells were defined as CD56+ / TCRa.p- cells. The antibody fusion proteins tested were Fuse 1147 (light grey), which consists of a knobs into holes bispecific antibody with a monovalent arm specific for human NKp30 (Fab) and a monovalent arm specific for human ROR1 (VHH), both located on the N-terminus of the knob (or hole). The R0R1 specific VHH was fused to the C-terminus of tire NKp30 specific LC. Fuse 1148 (dark grey) is identical to Fusel 147 expect drat it includes an NKp46 specific VHH on the C-tenninus of the CH3 domain (knob or hole). Fuse 1145 (black dots) is identical to Fusel 147 except that it includes an exemplary IL-18 variant on the N-terminus of the hole (or the knob if the NKp30 specific fab is on the hole) and IL- 15m 10-1 on the C-terminus of tire hole (or knob). Fuse 1146 (black stripes) is identical to Fusel 148 except that it includes an exemplary IL-18 variant on the N-terminus of the hole (or the knob ifthe NKp30 specific fab is on the hole) and IL-15mlO-l on the C-tenninus of the hole (or knob if tire NKp46 specific VHH is on the hole). Tirus, to simplify. Fusel 147, Fusel 18, Fusel 145 and Fusel 148 consist of NKp30XrorI, NKp30XrorlxNKp46, NKp30Xrorl-IL-18 / IL-15 and NKp30XrorlxNKp46-IL- 18 / LL- 15, respectively. Shown in FIG. 9A are flow cytometric dot plots of CD56 versus TCRap. A box has been drawn around the NK cells (CD56+ / TCRaP- cells) and the frequency of these cells is shown to the left of the box. The data is also shown graphically as a bar chart in FIG. 9B. The anti-HEL specific antibody (human IgGl LALA) was used as the negative isotype control. Relative to tire negative control and the engagers without the inclusion of cytokines, Fuse 1145 and Fusel 146 induced an increased frequency of NK cells by about 8.5 fold and 13.7 fold, respectively. We also measured tire total number of NK cells associated with the culture of PBMC with each test article (at 100 Pm). The fold expansion of NK cells derived from this data relative to that observed for anti-HEL is shown in FIG. 9C. No appreciable expansion was observed using engagers without the inclusion of cytokines, hr contrast, Fuse 1145 and Fusel 146 induced expansion of about 34 fold and 69 fold, respectively. The data indicates that addition NKp46 targeting to NKp30 targeted-IL-18 / IL-15 enhances the frequency and expansion ofNK cells from human PBMC.Example 9

[0460] As shown in figure 10, we assessed the agonist activity of y982 TCR specific VHH fused to the Fc domain ofhuman IgGl to induce redirected lysis of P815 cells stably transduced with Egfp and firefly luciferase (P815) by expanded y982 T cells. The assay design was as such: y982 T cells enriched / expanded from the PBMC of a healthy human donor was mixed with the P815 at an E:T ratio of 3: 1. After 24 hours, tumor cell cytotoxicity was assessed via luciferase activity (i.e. tumor cell viability). Non linear x-y plots of killing percentage as a function of test article concentration is shown is FIGs 10A-10B. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.Example 10

[0461] As shown in figure 11, we assessed the capacity of an exemplary IL- 15 variant and / or an exemplary? pro-IL18 targeted to y982 T cells via a Vv9V32 TCR x R0R1 bsAb cytokine fusion to induce y982 T cell mediated IFNy release and tumor cell killing. Tire assay design w as as such: y982 T cells enriched / expanded from the PBMC of a healthy human donor was mixed with the R0R1+ tumor cell. Jurkat stably expressing Egfp and human ROR1 at an E:T ratio of 3: 1. After 72 hours, tumor cell cytotoxicity was assessed via GFP fluorescence (i.e. tumor cell viability) and IFNy release was measured in tire supernatant using a standard ELISA protocol. The antibody fusion proteins tested were cytokine fusions to a Vy9V82 TCR x RORf bsAb (Fusel 139; black square) incorporating (1) IL-15mlO-l (Fusel 137 ; black triangle), (2) pro IL18mutA (Fusel 138 ; black reverse triangle) and (3) IL-15ml0-l and proIL18mutA (Fusel 136; open circle, dashed line). An anti-HEL (gray circle) was used as the isotype matched negative control. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 11 A) or IFNy release (FIG. 1 IB). Relative to Fusel 139, the incorporation of cytokmc(s) into the bsAb enhanced both tumor cell killing and IFNy release in the rank order of Fusel 136>Fusel 137>Fuseel 138 indicating that the combination of IL- 18 and IL- 15 was the most potent. The differential readout between test articles was more apparent for IFNy release compared to tumor cell killing. This is likely due to the high E:T ratio of 3 : 1 at which maximum tumor cell killing may be reached at a lower y962 T cell signaling threshold than IFNy release. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.Example 11

[0462] As show n in figure 12, we assessed the agonist activity of human NKp46 (FIGs 12A-12B) and human NKp30 (FIG. 12C) specific VHH fused to the Fc domain of human IgGl to induce redirected lysis ofP815 cells stably transduced w ith Egfp and firefly luciferase (P815) by expanded y982 T cells. The assay design was as such: Expanded NK cells enriched / expanded from the PBMC of a healthy human donor was mixed with the P815 at an E:T ratio of 1: 1 (FIG. 12A). 2: 1 (FIG. 12B) and 3: 1 (FIG. 12C). After 24 hours, tumor cell cytotoxicity was assessed via luciferase activity (i.e. tumor cell viability). Non linear x-y plots of killing percentage as a function of test article concentration is shown is FIGs 12A-12C.

[0463] Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.Example 12

[0464] As shown in figure 13, we assessed the capacity of an exemplary IL-15 variant and / or an exemplary pro-IL18 targeted to human NK cells via a NKp46 x R0R1 bsAb cytokine fusion to induce NK cell mediated IFNy release and tumor cell killing. The assay design was as such: NK cells enriched / expanded from the PBMC of a healthy human donor were mixed with tire R0R1+ tumor cell, Jurkat stably expressing Egfp and human R0R1 at an E:T ratio of 3:1. After 72 hours, tumor cell cytotoxicity’ was assessed via GFP fluorescence (i.e. tumor cell viability) and IFNy release was measured in tire supernatant using a standard ELISA protocol. The antibody fusion proteins tested were cytokine fusions to a NKp46 x ROR1 bsAb (Fusel 125; black square) incorporating (1) IL-15ml0-l (Fusel 127 ; black triangle), (2) pro IL18mutA (Fusel 126 ; black reverse triangle) and (3) IL-15ml0-l and pro IL18mutA (Fusel 124; open circle, dashed line). An anti-HEL (gray circle) was used as the isotype matched negative control. Shown are nonlinear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 13 A) or IFNy release (FIG. 13B). Relative to Fusel 125. the incorporation of cytokine(s) into the bsAb enhanced both tumor cell killing and IFNy release in the rank order of Fusel l24>Fusel 127>Fuseel l26 indicating that the combination of IL-18 and IL-15 was the most potent. The differential readout between test articles was more apparent for IFNy release compared to tumorcell killing. This is likely due to the high E:T ratio of 3: 1 at which maximum tumor cell killing may be reached at a lower NK cell signaling threshold than IFNy release. Summary tables of EC50, Emax and AUC (area under the curve) values are shown below each x-y plot.Example 13

[0465] As shown in figure 14, we assessed the capacity of an exemplary IL-15 variant and an exemplary pro-IL18 taigeted to human NK cells via a (1) human NKp30 x human R0R1 bsAb cytokine fusion (Fusel 147 and Fusel 145) or (2) NKp30 x NKp46 R0R1 bsAb cytokine fusion (Fusel 148 and Fusel 146), to induce NK cell mediated IFNy release and tumor cell killing. The assay design was as such: NK cells enriched / expanded from the PBMC of a healtiiy human donor were mixed with the ROR1+ tumor cell, Jurkat stably expressing Egfp and human ROR1 at an E:T ratio of 3: 1 . After 72 hours, tumor cell cytotoxicity was assessed via GFP fluorescence (i.e. tumor cell viability) and IFNy release was measured in the supernatant using a standard ELISA protocol. The antibody fusion proteins tested were a NKp30 x R0R1 bsAb (Fusel 147; black square) a NKp30 x R0R1 bsAb fused to IL-15ml0-l and pro IL18mutA (Fusel 145; open squares, dashed line), aNKp30 xNKp46 x RORl bsAb (Fusel 148; black circles) and a NKp30 x NKp46 x R0R1 bsAb fused to IL-15ml0-l and pro IL18mutA (Fusel 146; open circles, dashed line) . An anti-HEL (gray circle) was used as the isotype matched negative control. Shown are non-linear x-y plots of a titration of each test article as a function of tumor cell killing (FIG. 14) or IFNy release (FIG. 15). Relative to 1148, the incorporation of cytokine(s) into tire bsAb enhanced both tumor cell killing and IFNy release in tire rank order of Fusel 145>Fusel 146>Fuseel 147 indicating that (1) NKp30 x R0R1 was more potent than NKp30 x NKp46 x R0R1 and the combination of IL- 18 and IL-15 enhanced potency. Summary tables ofEC50, Emax and AUC (area under the curve) values are shown below7each x-y plot.Example 14Human peripheral blood mononuclear cell (PBMC) isolation

[0466] PBMCs were isolated from whole blood from healthy donors using Ficoll-Paque Plus medium. In brief, 35 Ml of diluted whole blood (1 volume of whole blood vs 1 volume of PBS) w as gently ovcrlayed on top of 15 Ml Ficoll-Paque Plus medium without disturbing the interface in a 50-M1 conical tube. After centrifuging for 40 minutes at 400 x g at room temperature without brake, the buffy coat (interface layer between Ficoll and serum) was collected and diluted in 5 volumes of PBS. After centrifuging for 5 minutes at 500 x g at room temperature, PBMCs were resuspended in PBS and washed once in PBS by centrifuging for 5 minutes at 500 x g at room temperature. PBMCs were then resuspended in 5 Ml of ACK lysis buffer and incubated for 5 minutes at room temperature to remove red blood cell residues. Afterthe 5-minute incubation period, 45 Ml PBS was added to PBMCs and centrifuged for 15 minutes at 100 x g at room temperature. At last, PBMCs were resuspended in culture medium (RPMI1640 with 10% heat-inactivated FBS and 1% peiticillin / streptomycin) for cytotoxicity and IFN gamma release assay set-up. Tostore PBMCs for further usage, PBMCs were pelleted down by centrifuging for 5 minutes at 500 x g at room temperature and then resuspended in freezing medium (90% heat-inactivated FBS and 10% dimethyl sulfoxide (DMSO)) for storage in liquid nitrogen.NK cell isolation and expansion

[0467] Fresh PBMCs were washed twice in PBS and resuspended in EasySep Buffer from StemCell at a density of 50 million cells per Ml in 50-M1 conical tube. 50 U1 of isolation cocktail from the NK cell isolation kit from StemCell was added to 1 Ml of cell suspension and incubated at room temperature for 5 minutes. After the 5-minute incubation period, 50 U1 of RapidSpheres from NK cell isolation kit was added to 1 Ml of the cell / antibody cocktail mixture. The total volume of the ccll / cocktail / bcad mixture was brought up to 25 or 50 Ml w ith EasySep Buffer and incubated at room temperature for fO minutes on EASYSEP magnet. After the 10-minute incubation period, the solution was transferred to a new 50-M1 tube by keeping the tube on the magnet and incubated at room temperature for 5 minutes on EASY SEP magnet. The solution with purified NK cell was collected. Freshly purified NK cells were used for expansion or frozen down and stored in liquid nitrogen for further usage.

[0468] To expand NK cells, lx 500 Ml of ImmunoCult™ NK cell expansion coating material fromImmunoCult NK cell expansion kit from StemCell was added to a non-tissue culture-treated 24-well plate and incubated at room temperature for 2 hours. During the 2-hour incubation, freshly isolated NK cells were resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at fire density of 1 million per Ml. After the 2-hour incubation, the plate was washed twice with PBS and 500 Ml of NK cell suspension (0.5 million NK cells) was added to one well of the coated 24-well plate. The plate was incubated at 37°C with 5% CO2 for 3 days and 500 Ml of ImmunoCult NK cell expansion medium was added to the well with NK cells. The platE was further incubated for another 4 days. At day 7, NK cells were collecteD by centrifuge at 300 x g for 10 minutes and resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at the density of 0.2 million per ML. 1 ML of collected NK cells (0.2 million NK cells) was added to a well of new coated 24- well plate and incubated for 4 days at 37°C with 5% CO2. At day 11, NK cells were collected again by centrifuge at 300 x g for 10 minutes and resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at the density of 0.2 million per ML. 1 ML of collected NK cells (0.2 million NK cells) was added to a well of new coated 24-well plate and incubated for another 3 days at 37°C ith 5% CO2. NK cells after 14-days’ expansion were collected and frozen down and stored in liquid nitrogen for further usage.T cell isolation

[0469] Fresh PBMCs were washed twice in PBS and resuspended in EasySep Buffer from StemCell at the density of 50 million cells per ML in 50-ML conical tube. 50 UL of isolation cocktail from T cell isolation kit from StemCell w as added to 1 ML of cell suspension and incubated at room temperature for 5 minutes. After the 5-minutes' incubation, 50 UL of RapidSpheres from T cell isolation kit was added to 1 ML of the cell / antibody cocktail mixture. The total volume of the ccll / cocktail / bcad mixture was brought up to 25 or 50 ML with EasySep Buffer and incubatedat room temperature for 10 minutes on EASYSEP magnet. After the 10-minutes’ incubation, the solution was transferred to a new 50-ML tube by keeping the tube on the magnet and incubated at room temperature for 5 minutes on EASYSEP magnet. The solution with purified T cell was collected. Freshly purified T cells were frozen down and stored in liquid nitrogen for further usage.Gamma 9 delta 2 T cell expansion

[0470] PBMCs were recovered from liquid nitrogen, washed tw ice in PBS and resuspended in culture medium (RPMI1640 with 10% heat-inactivated FBS and l%penicillin / streptomycin) at the density of 0.5 million cell per ML of medium. 5 UM IPP and 1000 U / ML IL-2 (final concentration) was added to the PBMCs and incubated for 7 days. At the end of 7-day incubation, tire percentage of gamma 9 delta 2 T cells was measured by flow cytometry, gamma 9 delta 2 T cells after 7-days' expansion were collected and frozen down and stored in liquid nitrogen for further usage.P815 redirected lysis assayOn tire day of setting up assay plates, collect P815 cell expressing EgfP and firefly luciferase and perform cell count via trypan blue exclusion on cell counter and adjust the density of the viable cell to 2xlOA5 cells / ML in complete RPMU640 medium with 10% heat-inactivated FBS, non-essential amino acid, Sodium pyruvate and I pen / strep. Add 50 UL target cell suspension to each well of a black clear flat-bottom 96-well plate using multichannel pipettor to result in a plating density of 10.000 cells / well. Prepare the testing article by serially diluting (3-fold, 5-fold or 8-fold serial dilution) in complete RPMU640 medium at the concentration of 4x final concentration and add 50 UL of 4x testing article preparation to the designated wells. After incubating the plate for 30 minutes at 37 °C with 5% CCE, collect the effector cells (expanded NK cell or expanded gamma 9 delta 2 T cell) to perform cell count via trypan blue exclusion on cell counter and adjust effector cells to desired density. Add 100 UL of effector cell suspension to P815 EgfP Flu cell with testing articles to bring tire final volume to 200 UL and briefly centrifuge the plate and incubate the plate for desired time length at 37 °C with 5% CO2. At tire end of incubation, remove plates from the incubator, spin for 30s at 300 x g, and remove 150ML of supernatant using a multichannel pipettor. Acclimate ONE-Glo Luciferase Assay solution from Promega to room temperature before use, add 50 UL of One-Gio solution to each well with cells, incubate for 2 min at room temperature and measure bioluminescence intensity (RLU). Killing percentage of target cells in each sample was obtained by using taiget cell alone group as 0% killing and PBMCs alone group as 100% killing [killing percentage of sample = (RLU of target cell alone— RLU of sample) / (RLU of taiget cell alone— RLU of PBMCs alone)* 100], Killing EC50 and Emax were then obtained by using built-in 4-parameter nonlinear regression cure fit in GRAPHPAD PRISM 8. Pelicluster CD3 antibody-primed T cell activation assay

[0471] Isolated T cells were recovered from liquid nitrogen and resuspended in RPMI1640 complete culture medium (RPMI1640 with 10% heat-inactivated FBS and 1% penicillin / streptomycin). The viability of cells was also detennined via trypan blue exclusion and the viable T cell density was adjusted to 1.33 million cells / ML in RPMI1640 complete culture medium. 75 UL of 0.67 million cells / ML T cell suspension (100,000 T cells) was added to the designated well in the 96-well plate. Pelicluster CD3 antibodv (mouse anti-human CD3 antibody clone CLB-T3 / 4.E1XE) was prepared in RPMI1640 complete culture medium at the concentration of 4 times of final concentration ad 75 UL of prepared pelicluster CD3 antibody was added to the cells in the designated well in the 96-well plate. FUSE proteins were prepared and serially diluted (5 -fold serial dilution) in RPMU640 complete medium from 200 NM to 2.56 PM. 150 UL of prepared FUSE proteins at different concentrations were then added to the designated wells in the 96-well plate with T cells and incubated for the dedicated time period (72 hours) at 37 °C with 5% CO2. At the end of the incubation period, the 96-well plates were centrifuged for 1 minute at 500 *g to transfer the supernatant to V-bottom storage plate using a multichannel pipettor for IFN gamma release assay.Cytotoxicity assay

[0472] On the day before assay setting up, selective antibiotics were removed from target cell (MDA-MB-231 EgfP FLUC cells). On the day of assay setting up, target cells were collected by brief TrypLE treatment and then washed with culture medium by centrifuging at 500 x g for 5 minutes at room temperature. Taiget cell lines were then resuspended in culture medium to determine the cell viability by trypan blue exclusion on Cellometer. The viable cell density was adjusted to 50,000 cells / ML in culture media. 100 UL target cell suspension (5,000 taiget cell) was carefully dispensed to each well of a 96-well black clear flat-bottom tissue culture plate using multichannel pipettor. The plate was then incubated for 4-5 hours in tissue culture incubator to ensure that the target cells have attached to the bottom of the 96-well plate.

[0473] Expanded NK cells or gamma 9 delta 2 T cells were pelleted down by centrifuge for 5 minutes at 500 x g at room temperature and resuspended in culture medium. The viability of cells was also determined via trypan blue exclusion and the viable NK or gamma 9 delta 2 T cell density was adjusted to 0.25 million cells / ML in RPMU640 medium with 10% FBS and 1% pcnicillin / strcptomycin. After 4-5 hours of incubation, culture medium was carefully removed from 96-well plates with taiget cells. 100 UL of 0.25 million cells / ML NK or gamma 9 delta 2 T cell suspension (25,000 cells) was added to the designated well in the 96-well plate with taiget cell, which would result in the E:T ratio of 5 : 1.

[0474] FUSE proteins were prepared and serially diluted (5-fold serial dilution) in RPM11640 medium with10% FBS and l%penicillin / streptomycin ranging from 200 NM to 2.56 PM. 100 UL of prepared FUSE proteins at different concentrations were then added to the designated wells in the 96-well plate with effector cells and taiget cells and incubated for the dedicated time period (48 hours) at 37 °C with 5% CO2. At the end of the incubation period, tire 96-well plates were centrifuged for f minute at 500 xg to transfer 50ML of supernatant to V-bottom storage plate using a multichannel pipettor for IFN gamma release assay. ONE-Glo luciferase Assay solution was brought to room temperature. 50 UL of One-Gio solution was then added to the designated well and incubated for 2 minutes at room temperature. The bioluminescence (IFU) was measured on a plate reader w ith preset Bio-luminance protocol. Killing of taiget cell was calculated by using the fonnula [Killing percentage of testing sample = (IFU of sample with PBMC and taiget cell— IFU of testing sample) / (IFU of sample with PBMC and target cell— IFU of sample with PBMC alone)* 100],IFN gamma release detection

[0475] IFN gamma release from cytotoxicity assay was measured using human IFN gamma ELISA detection kit. hr brief, coating antibody provided in the kit was diluted to suggested concentration by following the protocol provided by the kit manufacturer. 100 ul of diluted coating antibody was added to the Nunc MaxiSorp flatbottom 96-well plate. Plates were sealed and incubated overnight at 4 °C. On tire following day, tire plates were then washed 4 times with the Wash Buffer. To block non-specif...

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising a scaffold polypeptide and at least two of (a)-(c) : a. one or more of an interleukin 15 (IL- 15) variant, b. one or more of an interleukin 18 (IL-18), a fragment of the IL-18, an IL-18 variant, or a fragment of the IL- 18 variant, and c. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA).

2. The fusion protein of claim 1, wherein the one or more poly peptides capable of binding the TAA comprise a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH.

3. The fusion protein of claim 1 , wherein the TAA comprises EGFR HERL or DLL.

4. The fusion protein of claim 1, wherein the activation receptor comprises cluster of differentiation (CD) 3, CD 16, y9 TCR 82 TCR or 81 TCR or wherein the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1. NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40.

5. The fusion protein of claim 1, comprising one or more of an interleukin 15 (IL- 15) variant and one or more of an interleukin 18 (IL- 18), a fragment of the IL- 18. an IL- 18 variant.

6. The fusion protein of any one of claims 1-5, further comprising a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD16, y9 TCR 82 TCR or 81 TCR and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40.

7. The fusion protein of claim 1, wherein the IL-15 variant, the interleukin 18 (IL-18), the fragment of the IL-18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, are each independendy fused to the C-tenninus of the scaffold polypeptide.

8. The fusion protein of claim 1, wherein the IL- 15 variant, the interleukin 18 (IL- 18), the fragment of the IL- 18. the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, are each independendy fused to the N-terminus of the scaffold polypeptide.

9. The fusion protein of claim 1, wherein the scaffold polypeptide is an antibody, the IL-15 variant, the interieukin 18 (IL- 18), the fragment of the IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant, or the receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, are each independendy fused to any one of one of a C-terminus or an N-terminus of a heavy chain or a light chain, or fused to any one of a CH2 domain, or hinge region of the antibody.

10. The fusion protein of claim 1, wherein the scaffold polypeptide is an antibody or a fragment thereof.

11. The fusion protein of claim 10, wherein the antibody is an IgA, IgM, IgG, or IgE antibody.

12. The fusion protein of claim 10, wherein the antibody is an anti-PD-1 antibody or anti-PD-Ll antibody.

13. The fusion protein of claim 1, wherein the scaffold polypeptide is a fragment crystall izablc (Fc) region or a fragment thereof.

14. The fusion protein of claim 13, wherein the fiagment crystallizable (Fc) region is an Fc region from an IgG4. knobs-in-hole (KiH) Fc, or IgGl .

15. The fusion protein of claim 13, wherein the fragment crystal I izable (Fc) region is a knobs-in-hole (KiH) Fc.

16. The fusion protein of claim 1, wherein the scaffold polypeptide is a polypeptide or protein capable of translocating into an endoplasmic reticulum (ER), or a fragment thereof.

17. The fusion protein of claim 1. wherein the scaffold polypeptide is selected from fragment crystallizable (Fc) region, human serum albumin (HSA). beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof.

18. The fusion protein of any one of claims 1-17, wherein IL- 15 variant comprises the sequence of Formula I: SIH19. The fusion protein of any one of claims 1-18, wherein the IL-18 variant comprises20. Tire fusion protein of any one of claims 1-19, wherein the IL- 18, the fragment of IL- 18, tire IL- 18 variant, or tire fragment of the IL- 18 variant further comprises its propeptide (PP) or a PP variant, and optionally, wherein the PP or the PP variant is on tire N-temtinus end relative to tire IL- 18, the fragment of IL- 18, the IL- 18 variant, or the fragment of the IL- 18 variant.

21. The fusion protein of any one of claims 1-19, wherein the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL- 18 variant further comprises a short polypeptide or protein.

22. Tire fusion protein of any one of claims 1-21, wherein the fusion protein further comprises one or more cleavage sites and the fusion protein is cleaved attire one or more cleavage sites by one or more proteases.

23. The fusion protein of claim 22, wherein the one or more cleavage sites is between the IL- 18. a fragment of IL- 18. an IL- 18 variant, or a fragment of the IL- 18 variant and tire scaffold polypeptide, or within the PP, between PP or the PP variant and tire IL- 18, a fragment of IL- 18, an IL- 18 variant, or a fragment of the IL- 18 variant, or within the PP, between tire PP or the PP variant and the scaffold polypeptide, orwithin the IL- 18, a fragment of IL- 18, an IL- 18 variant or a fragment of the IL- 18 variant, or within the PP, or a combination thereof.

24. The fusion protein of any one of claims 2-23, wherein the R0R1 VHH comprises: a polypeptide having SEQ ID NO:325 (complementarity-determining region (CDR) 1 of 2A11). a polypeptide having SEQ ID NO:326 (CDR2 of 2A11), a polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination thereof; OR a variant of the polypeptide having SEQ ID NO:325 (CDR1 of 2A11), a variant of the polypeptide having SEQ ID NO:326 (CDR2 of 2A11), a variant of the polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or a combination thereof, wherein the variant of the polypeptide having SEQ ID NO:325 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:

325. wherein the variant of the polypeptide having SEQ ID NO:326 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:326, wherein the variant of the polypeptide having SEQ ID NO:327 comprises one or more deletions, additions or a substitutions of an amino acid residue in the polypeptide having SEQ ID NO:327, and wherein: the variant of the polypeptide having SEQ ID NO: 325 and the variant of the polypeptide having SEQ ID NO:327 do not replace cysteine residues in the polypeptide having SEQ ID NO:325 and the polypeptide having SEQ ID NO:327, or the variant of the polypeptide having SEQ ID NO: 325 and the variant of the polypeptide having SEQ ID NO:327 replaces one or both of the cysteine residues in the polypeptide having SEQ ID NO:325 and / or one or botii of the cysteine residues in the polypeptide having SEQ ID NO:327 with an amino acid that contains a cross-linking functional group.

25. A polynucleotide encoding a fusion protein of any one of claims 1-24.

26. An expression vector comprising the polynucleotide of claim 25.

27. A cell transfected with the expression vector of claim 26.

28. The cell of claim 27, wherein the cell is a mammalian cell.

29. The cell of claim 28, wherein the mammalian cell is a CHO cell or a HEK-293 cell.

30. The cell of claim 27, wherein tire cell is a bacterial cell or yeast cell.

31. A method of producing a fusion protein, comprising: culturing the cell of any one of claims 27-30, in cell culture medium to allow the fusion protein to be produced, and optionally secreted into the cell culture medium.

32. The mctirod of claim 31, further comprising isolating the fusion protein.

33. The method of claim 32, further comprising purifying the fusion protein.

34. A method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering a fusion protein of any one of claims 1-24, to a subject in need thereof.

35. The method of claim 34, wherein the subject has cancer.

36. A method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering a fusion protein of any one of claims 1-24. to a subject in need thereof.

37. The method of claim 36, wherein the disease or condition is cancer.

38. A method of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering at least two of the following to a subject in need thereof: a. interleukin 15 (IL- 15) variant or IL- 15 fusion protein, b. interleukin 18 (IL-18), a fragment of the IL-18, IL-18 variant, a fragment of the IL-18 variant, or an IL- 18 fusion protein, c. a multispecific antibody construct comprising i . a receptor tyrosine kinase-like orphan receptor 1 (R0R1 ) VHH, and ii. a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatoiy receptor expressed on an immune cell, wherein optionally the activation receptor comprises cluster of differentiation (CD) 3, CD16, y9 TCR, 82 TCR or 81 TCR, and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40, and d. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA)39. The method of claim 38, wherein tire subject has cancer.

40. A method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering at least two of the following to a subj ect in need thereof: a. interleukin 15 (IL-15) variant or IL-15 fusion protein, b. interleukin 18 (IL- 18), a fragment of the IL- 18, IL- 18 variant, a fragment of the IL- 18 variant, or an IL- 18 fusion protein, c. a multispecific antibody construct comprising i. a receptor tyrosine kinase-like orphan receptor 1 (R0R1) VHH, and ii. a receptor binding polypeptide capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell,wherein optionally the activation receptor comprises cluster of differentiation (CD)3, CD16, 9 TCR, 82 TCR or 81 TCR. and wherein optionally the costimulatory receptor comprises cluster of differentiation (CD) 137. CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, 0X40, CD40L, or CD40, and d. one or more receptor binding polypeptides capable of binding an activation receptor and / or a costimulatory receptor expressed on an immune cell, or one or more polypeptides capable of binding a tumor associated antigen (TAA).

41. Tire method of claim 40, wherein tire disease or condition is cancer.

42. An interleukin 15 (IL-15) variant comprising the sequence of Fonnula I:43.

50. Tire IL-15 variant of claim 42, wherein the IL-15 variant comprises an IL-15 variant listed in Table 1A or Table 3A.

51. A fusion protein, comprising the IL-15 variant of any one of claims 42-50; and a scaffold polypeptide.

52. The fusion protein of claim 51, wherein the IL-15 variant is fused to the C-terminus of the scaffold polypeptide.

53. The fusion protein of claim 51 or claim 52, wherein the scaffold polypeptide is an antibody or a fragment thereof.

54. The fusion protein of claim 53, wherein the antibody is an IgA. IgM, IgG. or IgE antibody.

55. The fusion protein of claim 53, wherein the antibody is an anti-PD-1 antibody or anti-PD-Ll antibody.

56. The fusion protein of claim 51 , wherein the scaffold polypeptide is a Fc region or a fragment thereof.

57. Tire fusion protein of claim 51, wherein the scaffold polypeptide is a Fc region or a fragment thereof and the scaffold polypeptide does not comprise an Fab.

58. The fusion protein of claim 56, wherein the Fc region is an Fc region from an IgG4, knobs-in-hole (KiH) Fc, or IgGl.

59. The fusion protein of claim 56, wherein tire Fc region is a knobs-in-hole (KiH) Fc.

60. The fusion protein of claim 51, wherein the IL-15 variant is fused to the knob of the KiH Fc.

61. The fusion protein of claim 51, wherein the IL- 15 variant is fused to the hole of the KiH Fc.

62. A polynucleotide encoding an IL-15 variant of any one of claims 42-50, or a fusion protein of any one of claims 51-61.

63. An expression vector comprising the polynucleotide of claim 62.

64. A cell transfected with the expression vector of claim 63.

65. The cell of claim 64, wherein the cell is a mammalian cell.

66. The cell of claim 65, wherein the mammalian cell is a CHO cell or a HEK-293 cell.

67. The cell of claim 64, wherein the cell is a bacterial cell or yeast cell.

68. A method of producing an IL-15 variant or a fusion protein, comprising: culturing the cell of any one of claims 64-67, in cell culture medium to allow the IL- 15 variant or the fusion protein to be produced, and optionally secreted into the cell culture medium.

69. The method of claim 68, further comprising isolating the IL- 15 variant or the fusion protein, or purifying the IL- 15 variant or the fusion protein.

70. A metiiod of activating, promoting differentiation or promoting expansion of T cells, B cells or natural killer (NK) cells, comprising administering an IL-15 variant of any one of claims 42-50, or an IL-15 fusion protein of any one of claims 51-61, to a subject in need tiiereof.

71. The method of claim 70, wherein the subject has cancer.

72. A method of ameliorating, inhibiting or treating a disease or condition in a subject in need thereof, comprising administering an IL-15 variant of any one of claims 42-50, or an IL-15 fusion protein of any one of claims 51-61, to a subject in need thereof.

73. The mediod of claim 72, wherein the disease or condition is cancer.

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