Single-cell and multi-cell fusion proteins and their uses

Abstract

This article describes single-cytokine and multi-cytokine fusion proteins, as well as the VHH cytokine fusion protein. Methods for producing these fusion proteins and their uses are also described. Methods for administering the fusion protein as a single composition to subjects in need are also included.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 472,939, filed June 14, 2023; U.S. Provisional Patent Application No. 63 / 472,972, filed June 14, 2023; U.S. Provisional Patent Application No. 63 / 527,948, filed July 20, 2023; U.S. Provisional Patent Application No. 63 / 530,784, filed August 4, 2023; and U.S. Provisional Patent Application No. 63 / 641,265, filed May 1, 2024, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0002] References to sequence lists This application contains a sequence list named "096034_000006WOPT_SequenceListing.xml" submitted in a computer-readable form, which has a size of 1,254,546 bytes and was created on June 13, 2024. The information contained in this computer-readable form is hereby incorporated by reference in its entirety. Technical Field

[0003] This invention relates to fusion proteins and their use in treating diseases such as cancer. Summary of the Invention

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

[0005] Various embodiments provide a fusion protein comprising a scaffold polypeptide and at least two of (a) to (c): a. One or more variants of interleukin-15 (IL-15), b. One or more of interleukin-18 (IL-18), fragments of IL-18, IL-18 variants, or fragments of IL-18 variants, and c. One or more receptor-binding polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs).

[0006] In various embodiments, one or more peptides capable of binding TAA may include receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH.

[0007] In various implementations, the TAA may include EGFR, HER2, or DLL.

[0008] In various embodiments, the activating receptor may include differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR or δ1 TCR, or the co-stimulatory receptor may include differentiation cluster (CD)137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L or CD40.

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

[0010] In various embodiments, the fusion protein may further comprise a receptor-binding polypeptide capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on an immune cell, wherein optionally, the activating receptor includes differentiation cluster (CD)3, CD16, γ9TCR, δ2 TCR, or δ1 TCR, and wherein optionally, the co-stimulatory receptor includes differentiation cluster (CD)137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40.

[0011] In various embodiments, the IL-15 variant, interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH may each independently fuse to the C-terminus of the scaffold polypeptide. In various embodiments, the IL-15 variant, interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH may each independently fuse to the N-terminus of the scaffold polypeptide. In various embodiments, the scaffold polypeptide is an antibody, and the IL-15 variant, interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH may each independently fuse to either the C-terminus or N-terminus of the heavy or light chain, or to the C-terminus of the antibody. H 2. Merge either the structural domain or the hinge region.

[0012] In various embodiments, the scaffold peptide may be an antibody or a fragment thereof. In various embodiments, the antibody may be an IgA, IgM, IgG, or IgE antibody. In various embodiments, the antibody may be an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0013] In various embodiments, the scaffold polypeptide may be a crystallizable fragment (Fc) region or a fragment thereof. In various embodiments, the crystallizable fragment (Fc) region may be an Fc region derived from IgG4, a knocks-in-hole (KiH) Fc, or IgG1. In various embodiments, the crystallizable fragment (Fc) region may be a knocks-in-hole (KiH) Fc.

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

[0015] In various embodiments, the scaffold peptide may be selected from crystallizable fragment (Fc) regions, human serum albumin (HSA), β2 microglobulin, transferrin, fragment antigen-binding regions (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, and their fragments.

[0016] In various implementations, IL-15 variants may contain a sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKE FLQSX 103 VHIVX 108 MFIX 112 TS, in which a.X1 can be any amino acid. b.X4 can be any amino acid. cX 22 It can be any amino acid. dX 30 It can be any amino acid. eX 41 It can be any amino acid. fX 45 It can be any amino acid. gX 49 It is any amino acid except V. hX 53 It is any amino acid except E. iX 64 It can be any amino acid. jX 67 It can be any amino acid. kX 68 It can be any amino acid. lX 79 It can be any amino acid. mX 93 It can be any amino acid. nX 103 It can be any amino acid. oX 108 It is any amino acid, and pX 112 It is any amino acid; (SEQ ID NO:533).

[0017] In various implementations, IL-18 variants may include: a1.MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250) has amino acid positions 37-193, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250, or a2.MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) has amino acid positions 37-193, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251, and optionally also includes amino acid substitutions at one or more of C74, C104, C112, and C164, each independently substituted with valine, alanine, or serine, or a3. Selected from IL-18 variants in Table 6B; and b1. Optionally, the one to five amino acid substitutions are one or more of the following: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; or M149V or M149I; or b2. Optionally, 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.

[0018] In various embodiments, IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant may also include its propeptide (PP) or a PP variant, and optionally, the PP or the PP variant is located at the N-terminus relative to IL-18, the fragment of IL-18, the IL-18 variant, or a fragment of an IL-18 variant.

[0019] In various embodiments, IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant may also comprise a short polypeptide or protein.

[0020] In various embodiments, the fusion protein may further include one or more cleavage sites, and the fusion protein may be cleaved at one or more cleavage sites by one or more proteases. In various embodiments, the one or more cleavage sites may be located: between IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant and the scaffold polypeptide; or within PP, between PP or a PP variant and IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; or within PP, between PP or a PP variant and the scaffold polypeptide; or within IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; or within PP; or combinations thereof.

[0021] In various implementations, ROR1 VHH may include: 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 The variant of the polypeptide having SEQ ID NO:325 (CDR1 of 2A11), the variant of the polypeptide having SEQ ID NO:326 (CDR2 of 2A11), the 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 substitutions of amino acid residues 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 substitutions of amino acid residues in the polypeptide having SEQ ID NO:326, and wherein the variant of the polypeptide having SEQ ID NO:327 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having SEQ ID NO:327, and in: The variants of the polypeptide having SEQ ID NO:325 and the variants of the polypeptide having SEQ ID NO:327 do not replace the 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 replace one or both cysteine ​​residues in the polypeptide having SEQ ID NO:325 and / or one or both cysteine ​​residues in the polypeptide having SEQ ID NO:327 with amino acids containing cross-linking functional groups.

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

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

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

[0025] Various embodiments provide a method for producing any of the aforementioned fusion proteins, the method comprising: culturing any of the aforementioned cells in a cell culture medium to allow the production of the fusion protein, and optionally secreting it into the cell culture medium. Various embodiments also provide a method for producing any of the fusion proteins described herein, the method comprising: culturing any of the aforementioned cells in a cell culture medium to allow the production of the fusion protein, and optionally secreting it into the cell culture medium. In various embodiments, the method may further include isolating the fusion protein. In various embodiments, the method may further include purifying the fusion protein.

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

[0027] Various implementation schemes provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering any of the aforementioned fusion proteins to the subject in need. Various implementation schemes provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering any of the fusion proteins described herein to the subject in need. In various implementation schemes, the disease or symptom may be cancer.

[0028] Various implementation schemes provide a method for activating or promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, the method comprising administering to a subject in need at least two of the following: a. Interleukin-15 (IL-15) variants or IL-15 fusion proteins, b. Interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, a fragment of the IL-18 variant, or an IL-18 fusion protein. c. A multispecific antibody construct comprising: i. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH, and ii. Receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells. Optionally, the activating receptor includes differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, and Optionally, the co-stimulatory receptors include differentiation cluster (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40, and d. One or more receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more peptides capable of binding to tumor-associated antigens (TAAs).

[0029] In various implementation schemes, the subject had cancer.

[0030] Various implementation schemes provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject in need at least two of the following: a. Interleukin-15 (IL-15) variants or IL-15 fusion proteins, b. Interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, a fragment of the IL-18 variant, or an IL-18 fusion protein. c. A multispecific antibody construct, wherein the multispecific antibody construct comprises: iii. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH, and iv. Receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells. Optionally, the activating receptor includes differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, and Optionally, the co-stimulatory receptors include differentiation cluster (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40, and d. One or more receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more peptides capable of binding to tumor-associated antigens (TAAs).

[0031] In various implementation schemes, the disease or symptom is cancer.

[0032] Various implementations provide a variant of interleukin-15 (IL-15) that comprises the sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKE FLQSX 103 VHIVX 108 MFIX 112 TS, in which a.X1 can be any amino acid. b.X4 can be any amino acid. cX 22 It can be any amino acid. dX 30 It can be any amino acid. eX 41 It can be any amino acid. fX 45 It can be any amino acid. gX 49 It is any amino acid except V. hX 53 It is any amino acid except E. iX 64 It can be any amino acid. jX 67 It can be any amino acid. kX 68 It can be any amino acid. lX 79 It can be any amino acid. mX 93 It can be any amino acid. nX 103 It can be any amino acid. oX 108 It is any amino acid, and pX 112 It is any amino acid; (SEQ ID NO:533).

[0033] In various implementations of the IL-15 variant a.X1 can be N (SEQ ID NO:534). b.X4 can be N (SEQ ID NO:535). cX 22 It could be D (SEQ ID NO:536). dX 30 It could be D (SEQ ID NO:537). eX41 It could be K (SEQ ID NO:538). fX 45 It could be L (SEQ ID NO:539). gX 64 It could be E (SEQ ID NO:540). hX 67 It could be I (SEQ ID NO:541). iX 68 It could be I (SEQ ID NO:542). jX 79 It can be N (SEQ ID NO:543). kX 93 It could be E (SEQ ID NO:544). lX 103 It could be F (SEQ ID NO:545). mX 108 It could be Q (SEQ ID NO:546), or nX 112 It can be N (SEQ ID NO:547), or Any two or more items from oa to o.

[0034] In various implementations of the IL-15 variant a.X1 can be N (SEQ ID NO:534). b.X4 can be N (SEQ ID NO:535). cX 22 It could be D (SEQ ID NO:536). dX 30 It could be D (SEQ ID NO:537). eX 41 It could be K (SEQ ID NO:538). fX 45 It could be L (SEQ ID NO:539). gX 49 It can be R or K (SEQ ID NO:548). hX 53 It can be G, K, I or A (SEQ ID NO:549). iX 64 It could be E (SEQ ID NO:540). jX 67It could be I (SEQ ID NO:541). kX 68 It could be I (SEQ ID NO:542). lX 79 It can be N (SEQ ID NO:543). mX 93 It could be E (SEQ ID NO:544). nX 103 It could be F (SEQ ID NO:545). oX 108 It could be Q (SEQ ID NO:546), or pX 112 It can be N (SEQ ID NO:547), or Any two or more items from qa to p.

[0035] In various implementations of the IL-15 variant a.X1 can be N or G (SEQ ID NO:550). b.X4 can be N, K, or L (SEQ ID NO:551). cX 22 It can be D or A (SEQ ID NO:552). dX 30 It can be D or N (SEQ ID NO:553). eX 41 It can be K or Q (SEQ ID NO:554). fX 45 It can be L or S (SEQ ID NO:555). gX 49 It can be R or K (SEQ ID NO:548). hX 53 It can be G, K, I or A (SEQ ID NO:549). iX 64 It can be E or Q (SEQ ID NO:556). jX 67 It can be I or T (SEQ ID NO:557). kX 68 It can be I or S (SEQ ID NO:558). lX 79 It can be N or Y (SEQ ID NO:559). mX93 It can be E or A (SEQ ID NO:560). nX 103 It can be F or L (SEQ ID NO:561). oX 108 It can be Q or T (SEQ ID NO:562), or pX 112 It is N or R (SEQ ID NO:563), or Any two or more items from qa to p.

[0036] In various implementation schemes, X 49 It can be R or K, and X 53 It can be G, K, I or A; (SEQ ID NO:564).

[0037] In various implementation schemes, X 30 It can be N (SEQ ID NO:565). In various implementations, X 64 It can be Q (SEQ ID NO:566). In various embodiments, X1 can be G (SEQ ID NO:567).

[0038] In various implementations, IL-15 variants may include the IL-15 variants listed in Table 1A or Table 3A.

[0039] Various embodiments provide an IL-15 fusion protein comprising any of the IL-15 variants described herein; and a scaffold peptide. Various embodiments provide an IL-15 fusion protein comprising any of the IL-15 variants described herein; and a scaffold peptide.

[0040] In various implementations, this IL-15 variant can be fused to the C-terminus of the scaffold peptide.

[0041] In various embodiments, the scaffold peptide may be an antibody or a fragment thereof. In various embodiments, the antibody may be an IgA, IgM, IgG, or IgE antibody. In various embodiments, the antibody may be an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0042] In various embodiments, the scaffold polypeptide may be an Fc region or a fragment thereof. In various embodiments, the scaffold polypeptide may be an Fc region or a fragment thereof, and the scaffold polypeptide does not contain Fab. In various embodiments, the Fc region may be an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1. In various embodiments, the Fc region may be a KiH (KiH) Fc. In various embodiments, an IL-15 variant may be fused with the pestle of a KiH Fc. In various embodiments, an IL-15 variant may be fused with the mortar of a KiH Fc.

[0043] Various embodiments provide a polynucleotide encoding any of the aforementioned IL-15 variants or any of the aforementioned IL-15 fusion proteins. Various embodiments provide a polynucleotide encoding any of the IL-15 variants or any of the IL-15 fusion proteins described herein.

[0044] Various embodiments provide an expression vector comprising any polynucleotide encoding any of the aforementioned IL-15 variants or any of the aforementioned IL-15 fusion proteins. Various embodiments provide an expression vector comprising any polynucleotide encoding any of the IL-15 variants or any of the IL-15 fusion proteins described herein.

[0045] Various embodiments provide a cell transfected with any expression vector containing any polynucleotide encoding any of the aforementioned IL-15 variants or any of the aforementioned IL-15 fusion proteins. Various embodiments provide a cell transfected with any expression vector containing any polynucleotide encoding any of the IL-15 variants or any of the aforementioned IL-15 fusion proteins. In various embodiments, the cell may be a mammalian cell. In various embodiments, the mammalian cell is a CHO cell or a HEK-293 cell. In various embodiments, the cell may be a bacterial cell or a yeast cell.

[0046] Various embodiments provide a method for generating any of the above-described IL-15 variants or IL-15 fusion proteins, the method comprising: culturing any of the above-described cell types in a cell culture medium to allow the generation of the IL-15 variant or fusion protein, and optionally secreting it into the cell culture medium. Various embodiments also provide a method for generating any of the IL-15 variants or IL-15 fusion proteins described herein, the method comprising: culturing any of the above-described cell types in a cell culture medium to allow the generation of the IL-15 variant or fusion protein, and optionally secreting it into the cell culture medium. In various embodiments, the method may further include isolating or purifying the IL-15 variant or fusion protein.

[0047] Various embodiments provide a method for activating or promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, comprising administering to a subject in need any of the aforementioned IL-15 variants or any of the aforementioned IL-15 fusion proteins. In various embodiments, the subject has cancer.

[0048] Various embodiments provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject any of the aforementioned IL-15 variants or any of the aforementioned IL-15 fusion proteins. Various embodiments provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject any of the IL-15 variants or any of the aforementioned IL-15 fusion proteins. In various embodiments, the subject has cancer.

[0049] Other features and advantages of the invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate various features of embodiments of the invention by way of example. Attached Figure Description

[0050] Exemplary embodiments are illustrated in the accompanying drawings. The embodiments and drawings disclosed herein are intended to be illustrative rather than restrictive.

[0051] Figures 1A to 1D Non-limiting examples of fusion proteins according to various embodiments of the present invention are depicted.

[0052] Figures 2A to 2DThe effects of an exemplary anti-PD1-pro-IL-18 antibody, one of three exemplary anti-PD1-mutant-IL-15 antibodies, or a combination of each of the anti-PD1-pro-IL-18 antibody and three mutant IL-15 antibody fusions on IFNγ release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (Pelicluster CD3, which is capable of cross-linking with CD3 and activating T cells without FcγR binding) for up to 72 hours are illustrated. For anti-PD1-pro-IL-18, an exemplary assay is Fuse694, which incorporates a pro-IL-18 (granulase B-cleavable IL18mutAS) into the C-terminal mortise or tenon of the IgG1-LALA version of nivolumab (Fuse691). For anti-PD1 mutant-IL-15, the three assays are Fuse765 (… Figure 2A Hollow triangle, dashed line), Fuse773 ( Figure 2B (Hollow triangle, dashed line) and Fuse774 ( Figure 2C (Hollow triangle, dashed line) These are obtained by incorporating an IL-15m4, IL15m9, or IL15m10 into the C-end pestle or mortar of Fuse691, respectively. Figures 2A to 2C This is a non-linear xy graph showing the relationship between test sample concentration and IFNγ release. The summation of EC50, Emax, and AUC (area under the curve) values ​​is shown in [the graph]. Figure 2D middle.

[0053] Figure 3 The effects of exemplary anti-PD1-pro-IL-18 antibody, exemplary anti-PD1-mutant-IL-15 antibody, and anti-PD1 antibody incorporating both the aforementioned exemplary pro-IL-18 and mutant IL-15 on IFNγ release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (PeliCluster CD3, which can crosslink with CD3 and activate T cells without FcγR binding) for 72 hours are illustrated. The summation of EC50, Emax, and AUC (area under the curve) values ​​is shown below the xy plot.

[0054] Figures 4A to 4B The ability of the NKG2D x ROR1 bispecific antibody (bsAb) with or without exemplary pro-IL18 and IL15 mutants to induce NK cell-mediated tumor cell killing and IFNγ release was demonstrated. Figure 4A and Figure 4BThese are nonlinear x-y plots showing the relationship between tumor cell killing and test sample concentration, and nonlinear x-y plots showing the relationship between IFNγ release and test sample concentration. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each x-y plot.

[0055] Figures 5A to 5B The effects of targeting NKG2D with exemplary IL-15 variants and exemplary pro-IL18 on NK cell expansion in PBMCs are shown. Figure 5A and Figure 5B The xy plots showing the percentage and number of NK cells measured on day 12 are presented as a function of titration concentration for each assay. 100 nM Fuse916 induced approximately 6-fold NK cell expansion compared to Fuse926.

[0056] Figures 6A to 6B The effects of targeting NKp46 with exemplary IL-15 variants and exemplary pro-IL18 on NK cell expansion in PBMCs are shown.

[0057] Figures 7A to 7B The ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via Vγ9Vδ2 TCR xROR1 bsAb cytokine fusions to induce γ9δ2 T cell proliferation in human PBMCs is demonstrated. Figure 7A and Figure 7B The xy plots showing the percentage and number of γ9δ2 T cells measured on day 12 are presented as a function of titration concentration for each assay. 100 nM of Fuse923 induced approximately 12-fold γ9δ2 T cell expansion relative to Fuse966.

[0058] Figures 8A to 8B The ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via the Vγ9Vδ2 TCR xROR1 bsAb cytokine fusion to induce γ9δ2 T cell-mediated IFNγ release and tumor cell killing is illustrated. Summarized EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0059] Figures 9A to 9C The effects of targeting NKp30 (with or without NKp46) with exemplary IL-15 and IL-18 variants on NK cells expanded from PBMCs are illustrated. The frequency and number of NK cells were measured after PBMCs from healthy human donors were incubated with 100 pM of the test sample described below for 21 days.

[0060] Figures 10A to 10B The agonist activity of γ9δ2 TCR-specific VHH fusion with the Fc domain of human IgG1 to induce redirected cleavage of expanded γ9δ2 T cells against P815 cells stably transduced with eGFP and firefly luciferase (P815) is shown. A non-linear xy plot of the percentage kill as a function of assay concentration is presented. The summation of EC50, Emax, and AUC (area under the curve) values ​​is shown below each xy plot.

[0061] Figures 11A to 11B This demonstrates the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via Vγ9Vδ2 TCR xROR1 bsAb cytokine fusions to induce γ9δ2 T cell-mediated IFNγ release and tumor cell killing. A non-linear xy-plot of tumor cell killing versus titration concentration for each assay is shown. Figure 11A ), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 11B The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0062] Figures 12A to 12C Human NKp46 (shown) Figures 12A to 12B ) and NKp30 ( Figure 12C A specific VHH fused with the Fc domain of human IgG1 to induce agonist activity in expanded γ9δ2 T cells to redirect and lyse P815 cells stably transduced with Egfp and firefly luciferase (P815). A non-linear xy plot of the percentage kill as a function of test sample concentration is shown. The summation of EC50, Emax, and AUC (area under the curve) values ​​is shown below each xy plot.

[0063] Figures 13A to 13B This demonstrates the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target human NK cells via NKp46 x ROR1bsAb cytokine fusions to induce NK cell-mediated IFNγ release and tumor cell killing. A non-linear xy-plot of tumor cell killing versus titration concentration for each assay is shown. Figure 13A ), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 13BCompared to Fuse1125, incorporating cytokines into bsAbs enhanced both tumor cell killing and IFNγ release, with the order being Fuse1124 > Fuse1127 > Fuse1126, indicating that the combination of IL-18 and IL-15 is most effective. The differences in IFNγ release readings between the test samples were more pronounced compared to tumor cell killing. This is likely due to the high E:T ratio of 3:1, at which maximum tumor cell killing is achieved when the NK cell signaling threshold is below IFNγ release. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0064] Figures 14 to 15 Exemplary IL-15 variants and exemplary pro-IL18 are shown to induce NK cell-mediated IFNγ release and tumor cell killing by targeting human NK cells with (1) human NKp30 x human ROR1 bsAb cytokine fusions (Fuse1147 and Fuse1145) or (2) NKp30 x NKp46 ROR1 bsAb cytokine fusions (Fuse1148 and Fuse1146). A nonlinear xy plot of tumor cell killing as a function of titration concentration for each assay is shown. Figure 14 ), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 15 The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0065] Figure 16 Exemplary fusion protein constructs with IL-15 variants according to various embodiments of the present invention are depicted.

[0066] Figures 17A to 17C The functional activities of the IL-15 variant associated with the following HEK-Blue IL-2 reporter cell induction and release of secretory embryonic alkaline phosphatase (SEAP) were depicted.

[0067] Figures 18A to 18B The functional activities of IL-15 variants associated with the following were described: T cell-mediated IFNγ release induced by PD1-targeted IL-15 variants in the presence of low “suboptimal” concentrations of anti-CD3.

[0068] Figure 19 The functional activities of IL-15 variants associated with the following were described: T cell-mediated IFNγ release induced by PD1-targeted IL-15 variants in the presence of allogeneic dendritic cells (DCs).

[0069] Figures 20A to 20MA diagram depicting the format of the pro-IL-18 fusion protein is provided. All proteins are incorporated into a human IgG scaffold, which is either wild-type homodimeric IgG1 or heterodimeric IgG1 Fc containing a club-and-mortar (KIH). When Pro-IL-18 is included in the fusion protein, it is fused in the following manner: (a) as a single cassette to the C-terminus of the club chain (or mortar chain); or (b) as a single cassette to the C-terminus of both the club chain and the mortar chain. Cleavage sites present in the pro-IL-18 cassette are indicated by a star. When a tumor-associated antigen-binding domain (TAA) is present, this domain is fused in the single cassette to the N-terminus of the club chain (or mortar chain) of the KIH heterodimeric IgG Fc, or to the N-terminus of both the club chain and the mortar chain. The TAA-binding domain can be VHH (e.g., EGFR-specific clone 9G8) or fab (e.g., EGFR-specific clone C225 from cetuximab).

[0070] Figures 21A to 21B The functional activities associated with the following were described: antibody-dependent cytotoxicity induced by FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1) and FUSE686 (single-arm anti-EGFR VHH 9G8-IgG1-pro-IL-18). Figure 21A ) and IFNγ release ( Figure 21B ).

[0071] Figures 22A to 22B The functional activities associated with the following were described: antibody-dependent cytotoxicity induced by FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1, targeting both EGFR and FcγR), or by a combination of FUSE556 and FUSE422 (IgG1-pro-IL-18, targeting FcγR only). Figure 22A ) and IFNγ release ( Figure 22B ).

[0072] Figure 23 The functional activities associated with the following were described: IFNγ release induced by FUSE691 (nivolumab), FUSE645 (EGFR-targeted IgG1-pro-IL-18, which was non-targeted in this system), FUSE694 (nivolumab-pro-IL-18), and recombinant human IL-18 in T cell / allogeneic DC co-cultures.

[0073] Figures 24A to 24BThe antibody-dependent cytotoxicity of FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1) and FUSE516 (single-arm anti-EGFR VHH 9G8-IgG1-pro-IL-18) is depicted. Illustrations illustrating the killing effect of PBMCs from each of four normal human donors on EGFR+ MDA-MB-231 tumor cells are shown in the figure. Figures 24A to 24B middle.

[0074] Figures 25A to 25B The functional activity associated with the following was described: antibody-dependent cytotoxicity induced by the pro-IL-18 fusion protein after 48 hours of co-culturing PBMCs with EGFR+ MDA-MB-231 tumor cells. Figure 25A ), IFNγ release ( Figure 25A Granzyme B release ( Figure 25B ) and the release / consumption of IL-18 ( Figure 25B The pro-IL-18 fusion proteins tested were FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1) and FUSE516 (single-arm anti-EGFR VHH 9G8-IgG1-pro-IL-18).

[0075] Figures 26A to 26B The functional activities associated with the following were described: FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1), a pro-IL-18 variant fused to the C-terminus of FUSE566 (with different protease cleavage sites between the Fc domain and pro-IL-18), were described. Figure 26A ), and variants containing the PGLALA mutation in the Fc domain to eliminate targeting of FcγR (FUSE627, Figure 26B Antibody-dependent cytotoxicity induced by granzyme A (FUSE658), granzyme B (FUSE516), MMP2 / 9 (FUSE659), and combinations of granzyme A, granzyme B, and MMP2 / 9 (FUSE660) were tested.

[0076] Figures 27A to 27B The functional activity associated with the following was described: antibody-dependent cytotoxicity induced by the pro-IL-18 fusion protein after co-culturing expanded NK cells with EGFR+ MDA-MB-231 tumor cells at an E:T ratio of 1:1 to 1:5 for 48 hours. Figure 27A ) and IFNγ release ( Figure 27BThe pro-IL-18 fusion proteins tested were FUSE556 (single-arm anti-EGFR VHH 9G8-IgG1) and FUSE516 (single-arm anti-EGFR VHH 9G8-IgG1-pro-IL-18).

[0077] Figures 28A to 28B The functional activity associated with the following was described: antibody-dependent cytotoxicity induced by the pro-IL-18 fusion protein after co-culturing expanded NK cells with EGFR+ MDA-MB-231 tumor cells at an E:T ratio of 2:1 for 48 hours. Figure 28A ) and IFNγ release ( Figure 28B The pro-IL-18 fusion protein tested was FUSE555 (bi-arm anti-EGFR FabC225-IgG1-pro-IL-18), which was compared with cetuximab.

[0078] Figures 29A to 29B The functional activity associated with the following was described: antibody-dependent cytotoxicity induced by the pro-IL-18 fusion protein after co-culturing expanded NK cells with HER2+ T47D tumor cells at an E:T ratio of 5:1 for 48 hours. Figure 29A ) and IFNγ release ( Figure 29B The pro-IL-18 fusion proteins tested were FUSE624 (bi-arm anti-HER2 Fab IgG1-pro-IL-18) and trastuzumab (Herceptin).

[0079] Figure 30 The ability of an anti-PDL1 antibody incorporating a pro-IL-18amut2 variant, designed to be unclecolytic by granzyme B, to induce T cell-mediated IFNγ release is shown. A summary of EC50, Emax, and AUC (area under the curve) is shown below the xy-plot.

[0080] Figures 31A to 31B The ability of an anti-PD1 antibody incorporating one of two pro-IL-18 variants (pro-IL18amut2 or pro-IL18amut9) designed to be cleaved by granzyme B was evaluated to assess the antibody's ability to induce T cell-mediated tumor cell killing. Figure 27A ) and the ability of IFNγ to release ( Figure 27B The summaries of EC50, Emax, and AUC (area under the curve) are shown below each xy plot.

[0081] Figure 32The ability of multiple IL-18 variants to induce SEAP-based non-targeted trans-release in HEK-Blue-IL-18 cells was demonstrated.

[0082] Figures 33A to 33B This study demonstrates the ability of multiple IL-18 variants targeting human PD-1 to induce cis-release-based IFNγ, derived from human PBMCs from healthy human donors. Figure 29A ) or human T cells ( Figure 29B ).

[0083] Figures 34A to 34B This demonstrates the ability of an exemplary IL-18 variant targeting mouse PD-1 to mediate tumor growth inhibition in an immune-active mouse tumor model of invasive syngeneic melanoma.

[0084] Figure 34C The number of different lymphocyte subsets per gram of tumor is shown in tumor-bearing mice treated with PBS (light gray), anti-PD1 (black), and Fuse1113 (horizontal line).

[0085] Figure 34D The frequencies of total bone marrow cells (CD11b+ cells), M2 macrophages (CD11b+ F4-80+ CD11c-CD206+ cells) and the following ratios were depicted: CD8+ T cells versus total myeloid-derived suppressor cells (MDSCs; CD11b+ Ly6C+ Ly6G- [M-MDSCs] and CD11b+ Ly6C-mid Ly6G+ [G-MDSCs]), CD8+ T cells versus M2 macrophages, anti-PD-1 responsive CD8+ T cells versus MDSCs, and CD8+ T cells versus CD4+ T cells.

[0086] Figures 35A to 35B This demonstrates the ability of an exemplary IL-18 variant targeting mouse PD-1 to mediate tumor growth inhibition in syngeneic colorectal cancer tumors in a fully immune-active mouse tumor model.

[0087] Figures 36A to 36B This demonstrates the ability of an exemplary IL-18 variant targeting mouse PD-1 to mediate tumor growth inhibition in syngeneically rapidly growing colorectal cancer tumors in a fully immune-active mouse tumor model.

[0088] Figure 37A The use of MC38i for subcutaneous injection in C57BL / 6 mice is shown. Serum was collected at 0, 24, 72, and 144 hours after treatment on day 0, and mouse IFNγ was measured.

[0089] Figure 37BThis study demonstrates C57BL / 6 mice that underwent re-attack with MC38i tumors after 90 days and experienced complete remission (CR) after treatment with αPD1-HT18cis or αPD-1-IL-18.

[0090] Figure 38 The images show BALB / c mice that underwent re-attack with CT26.C in the left flank at 65 days and re-attack with parental CT26 in the right flank at 70 days, and that experienced complete remission (CR) after treatment with αPD1-HT18cis. As a comparison, a group of five tumor-free mice received equivalent tumor inoculation.

[0091] Figure 39 This demonstrates the subcutaneous injection of B16-F10R into C57BL / 6 mice. When the average tumor volume was 75 mm... 3 Up to 100mm 3 During this period, mice were randomly divided into groups of 5 and treated with PBS, αPD-1, and αPD1-HT18cis at 15 mg / kg on days 0, 3, and 6. After 60 days, mice that had experienced CR after treatment with αPD1-HT18cis were challenged again with B16-F10.

[0092] Figure 40 C57BL / 6 mice with B16-F10R tumors were treated on days 0 and 3. Mice were euthanized on day 5, and tumors were collected and dispersed into single-cell suspensions for flow cytometry evaluation of TILs (CD45+ cells). TEM and TCM were defined as CD44+ / CD62L- and CD44+ / CD62L- cells within the CD8+ pool, respectively. αPD-1 responsive CD8+ T cells were defined as PD1+ / TCF1+ cells within the CD8+ pool.

[0093] Figure 41 C57BL / 6 mice with B16-F10R tumors were treated on days 0 and 3. Mice were euthanized on day 5, and tumors were collected and dispersed into a single-cell suspension for flow cytometry evaluation of TILs (CD45+ cells). All myeloid subsets were gated on CD11b. In this pool, MDSCs were defined as Ly6G+ / Ly6Cmid+Ly6G- / Ly6Chigh, and M2 macrophages were defined as F480+ / CD206+.

[0094] Figure 42A and Figure 42BThe effects of exemplary anti-PD1-pro-IL-18 antibodies containing different IL-18 variants on PBMC-mediated IFNγ release by activation with PeliCluster CD3 for 48 hours, washing, and re-exposure to 10-fold lower concentrations of PeliCluster CD3 for an additional 48 hours were depicted.

[0095] Figure 43 (Figures A through E) depict an exemplary fusion protein in which the N-terminus of pro-IL-18 is fused to the C-terminus of the club of a club-and-socket heterodimer IgG1 protein; the fusion protein has a structure from the N-terminus to the C-terminus comprising club-and-socket (KiH)Fc-propeptide (PP)-enterokinase-cleavable site (EK)-IL-18 wild-type or a variant thereof. This depicts exemplary fusion proteins in Table 6, such as ID: FUSE-480, FUSE-481, and FUSE-442. Pro-IL-18 is further modified to reduce molecular aggregation, wherein each cysteine ​​residue in both the propeptide and mature IL-18 is replaced with a serine (as denoted as "IL-18AS" in FUSE-480), an alanine (as denoted as "IL-18AA" in FUSE-481), or a valine (as denoted as "IL-18AV" in FUSE-442). Alternatively, the N-terminus of pro-IL-18 can be fused to the C-terminus of the salina chain of the KiH heterodimeric IgG1 protein. Sub-Figure E illustrates the biological activity of each compound, defined as EC50-SEAP.

[0096] Figure 44 (Figures A through E) depict an exemplary fusion protein in which the N-terminus of proIL-18 is coupled to the IgG1 CH3 domain (which is also as shown in Figures E). Figure 43The proIL-18 heterodimer IgG1 protein is fused to the C-terminus of the club chain, and four amino acid substitutions are incorporated into proIL-18. These substitutions are presumably designed to reduce binding to IL-18BP while maintaining wild-type binding to the IL-18 receptor complex, and are denoted as "pro-IL-18mut2". These fusion proteins have a structure from the N-terminus to the C-terminus containing the club chain (KiH)Fc-propeptide (PP)-enterokinase-cleavable site (EK)-IL-18mut2. Further modifications to pro-IL-18mut2 to reduce molecular aggregation involved substituting each cysteine ​​residue in both the propeptide and mature IL-18mut2 with serine (as denoted as "IL-18mut2AS" in FUSE-422; Figure B), alanine (as denoted as "IL-18mut2AA" in FUSE-423; Figure C), or valine (as denoted as "IL-18mut2AV" in FUSE-424; Figure D). Figure E illustrates the bioactivity of each compound defined as EC50-SEAP.

[0097] Figure 45 (Figures A through D) depict exemplary fusion proteins with or without a propeptide (Figure A) to examine the effect of the propeptide on masking the bioactivity of “IL-18AV” (Figure C), where Fc fusion variants were generated by incorporating “IL-18AV” with or without a propeptide (Figure A; FUSE-442) or without a propeptide (Figure B; FUSE-505). Figure D illustrates the bioactivity of each compound, defined as EC50-SEAP.

[0098] Figure 46(Figures A through D) depict exemplary fusion proteins with or without a propeptide (Figure A) to examine the effect of the propeptide on masking the biological activity of “IL-18mut2AV”, where Fc fusion protein variants were generated by incorporating either “IL-18mut2AV” without a propeptide (i.e., a mutated mature IL-18, denoted as “matIL-18mut2-AV”, see Figure B; FUSE-441) or with a propeptide (Figure A; FUSE-424). For fusion proteins lacking a propeptide, the EK cleavage site replacing the caspase 1 site was moved to a position directly between the CH3 domain of the thallium and the mature IL-18AV, without the addition of a flexible linker. Subplot C depicts activation readings obtained using HEK-Blue IL-18AV reporter cells after exposure to different concentrations of FUSE-441 (Fc-EK-IL-18AV) or FUSE-424 (Fc-EKpp-IL-18AV) with or without EK treatment. Subplot D shows the biological activity of each compound, defined as EC50-SEAP.

[0099] Figure 47 (Figures A through D) depict exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of either IgG1 Fc or IgG4 Fc protein; the fusion protein has a structure from the N-terminus to the C-terminus comprising IgG1 Fc propeptide (PP)-IL-18AV (FUSE-507; Figure A) and IgG4 Fc propeptide (PP)-IL-18AV (FUSE-509; Figure B). Figure C depicts activation readings using the HEK-Blue IL-18AV reporter cell assay, which exposes cells to different concentrations of FUSE-507 or FUSE-509 with or without EK treatment. Figure D illustrates the biological activity of each compound, defined as EC50-SEAP.

[0100] Figure 48 (Figures A through E) depict exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of an HSA with or without a propeptide (PP); the fusion protein has a structure from the N-terminus to the C-terminus comprising HSA-propeptide (PP)-IL-18AV (FUSE-501; Figure A) and HSA-IL-18AV (FUSE-503; Figure B). Figures C and D depict activation readouts. Figure E shows the biological activity of each compound, defined as EC50-SEAP.

[0101] Figure 49(Figures A through E) depict exemplary fusion proteins in which the N-terminus of pro-IL-18mut2 is fused to the C-terminus of an HSA with or without a propeptide (PP); the fusion protein has a structure from the N-terminus to the C-terminus comprising HSA propeptide (PP)-IL-18mut2AV (FUSE-502; Figure A) and HSA-IL-18mut2AV (FUSE-504; Figure B). Figures C and D depict activation readouts. Figure E shows the biological activity of each compound, defined as EC50-SEAP.

[0102] Figure 50 (Figures A through D) depict exemplary fusion proteins in which the C-terminus of pro-IL-18 is fused to the N-terminus of a mordone heterodimer IgG1 protein with or without a propeptide (PP); the fusion protein has a structure from the N-terminus to the C-terminus comprising propeptide (PP)-IL-18AV-mordone (KiH)Fc (FUSE-499; Figure A) and IL-18AV-mordone (KiH)Fc (FUSE-500; Figure B). Figure C depicts activation readouts. Figure D shows the biological activity of each compound defined as exposed or unexposed to caspase 1 at EC50-SEAP.

[0103] Figure 51A and Figure 51D Exemplary fusion proteins with structures from the N-terminus to the C-terminus are depicted: Fc-ppMMP2 / 9-cleavage site-IL-18-AV (FUSE-486) ​​or Fc-ppMMP9 / 2-cleavage site-IL-18-AV (FUSE-487), wherein these cleavage sites are specific to the metalloproteinases MMP2 and MMP9, with the preferred enzyme on the left side of the slash, or FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV). Figure 51B Activation readings associated with FUSE-486 and FUSE-487 with and without MMP2 treatment are depicted. Figure 51B The bioactivity of FUSE-486 and FUSE-487, defined as EC50-SEAP, is shown with or without MMP2 treatment. Figure 51CThe results showed that FUSE587 was attenuated to approximately 1 / 3,000 of recombinant human IL-18. Interestingly, we observed that cleavage of FUSE587 with MMP2 released an IL-18AV variant, which was still attenuated to approximately 1 / 100 of recombinant IL-18. In contrast, cleavage with granzyme B released an IL-18AV variant with similar activity to recombinant IL-18. Cleavage with granzyme B released mature IL-18AV without any N-terminal residue overhangs; while cleavage of FUSE486 and FUSE587 with MMP2 left N-terminal polypeptide overhangs of 11 and 15 amino acids, respectively. We hypothesize that these overhangs may attenuate IL-18AV activity, although to a lesser extent than the full-length variant propeptide. Figure 53 and Figure 55 This phenomenon was further studied in China. Figure 51D and Figure 51G Exemplary fusion proteins with structures from the N-terminus to the C-terminus were also described: Fc-ppGb-cleavage site-IL-18-AV (FUSE-485; B-19D) or Fc-ppGb-cleavage site-IL-18mut2-AV (FUSE-462; 51D) or Fab-cetuximab-Fc-ppGb-cleavage site-IL-18-AV (FUSE-517; 51G), wherein the cleavage site is specific to granzyme B (Gb). Figure 51E Activation readings associated with FUSE-462 and FUSE-485 were depicted with and without granzyme B treatment. Figure 51F The bioactivity of FUSE-462 and FUSE-485, defined as EC50-SEAP, is shown with or without granzyme B treatment. Figure 51H The activation readings associated with FUSE-517 were depicted with and without enzyme treatment. Figure 51I The bioactivity of FUSE-517, defined as EC50-SEAP, is shown with or without granzyme B treatment.

[0104] Figure 52 (Figures A through G) depict the effects of IL-18BP on the bioactivity of recombinant human IL-18 (rhIL-18) and on the bioactivity of the EK cleavage products of the exemplary fusion proteins Fc-ppEK-IL-18-AV (FUSE-442) and Fc-ppEK-IL-18mut2AV (FUSE-424). Figures B, D, and F show the bioactivity of each compound defined as EC50-SEAP with and without the addition of IL-18BP (competitive assay), respectively; Figures C, E, and G show the corresponding bioactivity defined as EC50-SEAP, respectively.

[0105] Figure 53 (Figure A) depicts an exemplary fusion protein in which the C-terminus of pro-IL-18 is fused to the N-terminus of the club of the club-shaped heterodimer IgG1 protein, and peptides of different sizes are fused to the N-terminus of mature IL-18. Figure 53 (Figure B) depicts the bioactivity of each fusion protein using the IL-18 reporter cell line HEK-Blue IL-18.

[0106] Figure 54A Figure 54B(i), Figure 54B(ii) Figure 54C Figures 54D(i), 54D(ii), and Figure 54E Human IL-18 engineered mutant fusion proteins according to various embodiments of the present invention are described.

[0107] Figure 55 The effect of the size of the peptide fused to the N-terminus of mature IL-18 on the bioactivity of a single IL-18AV fused to the N-terminus of IgG1 Fc was shown.

[0108] Figures 56A to 56H The effects of substitution of cysteine ​​residues in the propeptide and in mature IL18 (which fuse together to form a pro-IL-18 variant cassette) on the bioactivity of each variant are shown using the HEK Blue IL18 assay system.

[0109] Figures 57A to 57B The effects of targeting pro-IL18 to a region closely adjacent to its receptor complex (i.e., "cis activity") are shown.

[0110] Figures 58A to 58E A schematic diagram depicts various formats of anti-ROR1 / CD3 bispecific antibodies (also known as T-cell adaptors (TCEs)). The diagram illustrates the protein design of a TCE fusion protein comprising a kilometreotype (KIH) heterodimer IgG1 Fc scaffold, which has (1) a kilometreotype chain in the KIH (shown in the diagram). Figure 58A ,point Figure 58B ,point Figure 58D and points Figure 58E (middle) or mortar chain (shown in the middle) Figure 58C (a) a CD3-specific fragment antigen-binding region (Fab) on the N-terminus of a tandem tarsal chain, and (2) one or more VHHs or single-chain variable fragments (scFVs) specific for tumor-associated antigens (TAAs) (e.g., ROR1) at one or more locations. The illustrated examples show TAA binders linked to: (a) the N-terminus of a tandem tarsal chain (… Figure 58A(b) The N-terminus of the tarsal chain and the C-terminus of the CD3-specific (anti-CD3) Fab light chain ( Figure 58B (c) The N-terminus of the Fab chain and the C-terminus of the CD3-specific (anti-CD3) Fab light chain ( Figure 58C (d) The N-terminus and C-terminus of the mortise chain ( Figure 58D ), and (e) only the N-terminus of the mortise chain ( Figure 58E ).

[0111] Figures 59A to 59B The yield of an exemplary fusion protein (denoted as FUSE ID, "FUSE-XXX") is shown. Figure 59A ) and melting temperature (Tm) (2B).

[0112] Figures 60A to 60D The epigenetic binding affinity of exemplary TCEs to CD3 expressed on the cell membrane of human T cells and non-human primate (NHP) cynomolgus monkey T cells is shown.

[0113] Figures 61A to 61E The epigenetic binding affinity of exemplary TCEs to ROR1 expressed on the cell membrane was depicted using a series of engineered MDA-MB-231 variant cell lines expressing ROR1 at different cell surface densities. Figures 61F to 61K depict the ability of exemplary TCEs specific for ROR1 to bind to the distal membrane ROR1 Ig domain.

[0114] Figures 62A to 62D The binding affinity (KD) of the following TCEs to ROR1, determined by biomembrane interferometry (BLI), was described: FUSE-211 ( Figure 62A ), FUSE-393 ( Figure 62B ) and FUSE-394 ( Figure 62C The binding affinity calculated for them is as follows: Figure 62D As shown.

[0115] Figures 62E to 62G The 2A11 VHH-Fc fusion protein (in) was depicted Figure 62E The protein is referred to as FUSE-112 in the middle and the 2A11 VHH mutant-Fc fusion protein (in the middle). Figure 62F The binding affinity of FUSE-453 to ROR1 is represented in the figure, where the binding affinity calculated for them is shown in the figure. Figure 62G This indicates that replacing cysteine ​​residues in CDR1 and CDR3 of ROR1-specific VHH would have an effect.

[0116] Figures 62H to 62J The 5A1 VHH-Fc fusion protein (in) was depicted Figure 62HThe binding affinity of the 5A1VHH mutant-Fc fusion protein (represented as FUSE-179 in Figure 64) to ROR1 is shown in Figure 64, where the calculated binding affinity for them is shown in Figure 65. Figure 62J This indicates that replacing cysteine ​​residues in CDR1 and CDR3 of ROR1-specific VHH would have an effect.

[0117] Figures 63A to 63D This study demonstrated the induction of TCEs (characterized as percentage of death) of ROR1-dependent cytotoxicity in human peripheral blood mononuclear cells (PBMCs) mediated by MDA-MB-231 tumor cells, where the tested TCEs included FUSE-211, FUSE-393, and FUSE-394, and the human PBMCs were derived from donor 1 ( Figure 63A ), donor 2 ( Figure 63B ) or donor 3 ( Figure 63C ). Figure 63D The EC calculated when each TCE induces a 50% tumor cell killing percentage was summarized. 50 .

[0118] Figure 63E Figure 6H illustrates the TCE induction of IFN-γ release from human PBMCs in the presence of MDA-MB-231 tumor cells (characterized as the concentration of IFN-γ in the cell culture supernatant). The TCEs tested included FUSE-211, FUSE-393, and FUSE-394, and the human PBMCs were derived from donor 1 (…). Figure 63E ), donor 2 ( Figure 63F ) or donor 3 ( Figure 63G ). Figure 63H The EC50 calculated when the maximum IFN-γ release induced by each TCE reaches 50% is summarized. 50 .

[0119] Figures 63I to 63O This demonstrates that, compared to FUSE-277 (a typically highly effective TCE that serves as a “non-uncoupling” control TCE), FUSE-211, FUSE-393, and FUSE-394, while maintaining cytotoxic efficacy (maximum killing of MDA-MB-231), mediate a reduction in the release of ROR1-dependent IFN-γ from human PBMCs (average of the three donors) or Pan-T cells, or in other words, “uncoupling cytotoxicity from cytokine release.”

[0120] Figures 63P to 63V The study depicted the ROR1-dependent killing, induction of cytokine release, and calculated uncoupling ratios after 24, 48, and 72 hours of co-culture of the PBMC effector with the ROR1+ MDA-MB-231 tumor target.

[0121] Figures 64A to 64C This study demonstrates TCE induction of ROR1-independent cytotoxicity in human PBMC-mediated T47-D tumor cells (which do not express ROR1), characterized by the concentration of IFN-γ in the co-culture supernatant. Sub-figures A through C show the results where the human PBMCs were derived from donor 1 (sub-figure A), donor 2 (sub-figure B), and donor 3 (sub-figure C), respectively.

[0122] Figures 65A to 65E and Figure 65G Figures 65K depict the TCE induction of ROR1-dependent cytotoxicity and cytokine release mediated by human PBMCs, with the induction varying with ROR1 cell surface density. Data were generated using a range of cell lines from different sources expressing varying amounts of ROR1 molecules on their cell surfaces. Cell lines are shown in descending order of ROR1 density on their cell surfaces. Specifically, NCCIT ( Figure 65A , Figure 65G ), MDA-MB-231 ( Figure 65B , Figure 65H ), NCI-H1975 ( Figure 65C , Figure 65I ), DU-145 ( Figure 65D , Figure 65J ) and ROR1 negative T-47D ( Figure 65E (Figure 65K). Figure 65F and Figure 65L The cytotoxicity and cytokine release efficacy induced by each TCE on a range of cell lines with different surface ROR1 densities were summarized. Figure 65M The calculated uncoupling ratios for each TCE for different cell lines are shown.

[0123] Figures 66A to 66E and Figures 66G to 66K The effects of TCE induction of ROR1-dependent cytotoxicity and cytokine release mediated by human PBMCs on a series of engineered MDA-MB-231 variants expressing different cell surface densities of ROR1 were described. The surface density of ROR1 on engineered MDA-MB-231 cells was approximately 2,000,000 molecules / cell (MDA++). Figure 66A , Figure 66G Approximately 500,000 molecules / cell (MDA+); Figure 66B , Figure 66H ), approximately 35,000 molecules / cell (MDA; Figure 66C , Figure 66I Approximately 17,000 molecules / cell (MDA-low); Figure 66D , Figure 66J ) and 0 molecules / cell (MDA-KO; Figure 66E , Figure 66K ). Figure 66F and Figure 66L The efficacy of each TCE in inducing cytotoxicity and cytokine release in MDA-MB-231 cells with different surface ROR1 densities was summarized. Figure 66M This represents the uncoupling ratio (cytotoxicity caused by cytokine release) calculated for each TCE, and Figure 66N This is a regression analysis of the IFNγ values ​​and ROR1 cell surface density in the non-uncoupled TCE control and the exemplary uncoupled TCE.

[0124] Figure 67A and Figure 67B The expression of TCE-induced activation markers CD69 and CD25 on T cells after co-culturing PBMCs with tumor cells expressing ROR1 was described. Figure 67C The potency of each TCE in inducing CD69 or CD25 was summarized and defined as EC. 50 Combine. Figure 67D and Figure 67F This is a bar graph illustrating the uncoupling between cytotoxicity and the induction of cell surface biomarkers CD69 and CD25 for T cell activation. Figure 67E and Figure 67G This is a bar graph illustrating the uncoupling between CD69 and CD25 induction and cytokine release.

[0125] Figures 68A to 68C The study described the effect of 5 mg / kg over a 10-day period. Figure 68A ) or 0.5 mg / kg ( Figure 68B The pharmacokinetics (PK) of an exemplary TCE administered at a single dose to wild-type C57BL / 6 mice. Figure 68C The PK parameters for each test item are described.

[0126] Figures 69A to 16 9E depicts two humanized mouse xenograft tumor models ( Figure 69A and Figure 69C A schematic diagram. In both cases, highly aggressive individuals will be ROR1. + Teratoma NCCIT was injected into immunodeficient mice. Tumor growth over time in the first model is shown in... Figure 69B In the second model, tumor growth over time is shown in... Figure 69D and Figure 69E In both cases, the test sample consisted of FUSE-394 and the uncoupling control TCE (FUSE-399).

[0127] Figures 70A to 70E The ability of exemplary TCE to induce T cell-mediated killing of the following cells: ROR1-positive tumor cells (MDA-MB-231); Figure 70A ROR1 negative tumor cells (T-47D); Figure 70B ), and a mixture of ROR1-positive and ROR1-negative tumor cells ( Figure 70C and Figure 70D For the latter, the killing effect on ROR1-positive tumors was shown to be... Figure 70C The killing effect on ROR1-negative tumors is termed "bystander killing," and its comparison with uncoupling TCE is shown in [the figure]. Figure 70D middle. Figure 70E The IFNγ produced by the same T cell used to assess “bystander killing” was depicted, varying with exemplary TCE concentrations.

[0128] Figures 71A and 71B depict (a) the percentage of ROR1-positive tumor cells (MDA-MB-231) killed as a function of T cell number in the context of an exemplary TCE (Figure 71A), and (b) TCE-mediated “continuous killing”, which was calculated as the average number of TCE-induced ROR1-positive tumor cells killed per T cell over a 24-hour period (Figure 71B).

[0129] Figure 72 The calculated epigenetic binding affinity (EC50 binding) of ROR1-specific VHH-Fc incorporated with the following terms is depicted: nonhumanized VHH clone 5A1 (FUSE179; black circle), humanized clone 5A1-FA9-HK1 (FUSE559; black square), or humanized clone 5A1-HK1 (FUSE524; black triangle).

[0130] Figures 73A and 73B depict the calculated affinity (KD) of ROR1-specific VHH-Fc incorporating the following: nonhumanized VHH clone 5A1 (FUSE179; Figure 73A), or humanized clone 5A1-FA9-HK1 (FUSE559; Figure 73B). Figures 74A to 74B The epigenetic binding affinity (EC50 binding) of two bispecific antibodies (bsAb) was described.

[0131] Figures 75A to 75B Several ROR1 x CD3-specific bsAbs were characterized for the following abilities: when co-mixed with ROR1+ MDA-MB-231 tumor cells, (1, Figure 75A ) induces T cell-mediated ROR1+ MDA-MB-231 tumor cell killing, and (2, Figure 75BIt induces T cell-mediated IFNγ release.

[0132] Figures 76A to 76I The following capabilities of several ROR1 x CD3-specific bsAbs were described: when co-mixed with the aforementioned tumor cells, (1, Figures 76A to 76D ) induces PBMC-mediated killing of four different tumor cell lines, and (2, Figures 76E to 76H It induces PBMC-mediated IFNγ release.

[0133] Figures 77A to 77F Several ROR1 x CD3-specific bsAbs were characterized for the following abilities: when co-mixed with ROR1+ MDA-MB-231 tumor cells, (1, Figure 77A ) induces PBMC-mediated ROR1+ MDA-MB-231 tumor cell killing, and (2, Figure 77B ) induces PBMC-mediated IFNγ release; (3, Figure 77C When co-mixed with ROR1+ MDA-MB-231 tumor cells, it induces upregulation of CD69 expression on the cell surface of T cells within PBMCs; (4, Figure 77D When co-mixed with ROR1+ MDA-MB-231 tumor cells, it induces upregulation of PD-1 expression on the cell surface of T cells within PBMCs; (5, Figure 77E When co-mixed with ROR1+ MDA-MB-231 tumor cells, it induces upregulation of TIGIT expression on the cell surface of T cells within PBMCs.

[0134] Figures 78A to 78C The following capabilities of FUSE399 (black inverted triangle) and FUSE608 (black circle) were described: inducing CD8+ T cell-mediated killing of a constant number (5000 cells) of ROR1+ MDA-MB-231 cells in the range of 313 to 40,000 CD8+ T cell numbers.

[0135] Figure 79A Figure 22D illustrates whether FUSE608 induces bystander killing in ROR1-negative tumors mixed with ROR1-positive tumors.

[0136] Figures 80A to 80B The ability of FUSE608 and the uncoupled benchmark ROR1 x CD3 bsAb (i.e., FUSE399) to mediate tumor growth inhibition (TGI) in a mouse tumor xenograft model was described. Detailed Implementation

[0137] All references cited in this article are incorporated herein by reference in their entirety as if listed in full. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced OrganicChemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (NewYork, NY 2013); and Sambrook and Russel, Molecular Cloning: A LaboratoryManual 4 th The book, ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides general guidance to those skilled in the art regarding many of the terms used in this application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2. nd ed. (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. Patent No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., 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):1126-36).

[0138] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used in the practice of this invention. In fact, this invention is by no means limited to the methods and materials described. For the purposes of this invention, the following terms are defined.

[0139] As used herein, when the terms “about” or “approximately” are used in conjunction with a referenced numerical indication, they mean the referenced numerical indication ± up to 5% of that referenced numerical indication, unless otherwise specifically specified herein. For example, the expression “about 50%” covers a range of 45% to 55%. In various embodiments, when the term “about” is used in conjunction with a referenced numerical indication, it may mean the referenced numerical indication ± up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numerical indication, unless otherwise specifically specified in the claims.

[0140] The percentage of sequence identity (%) relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. After sequence alignment and the introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, any conserved substitutions as part of the sequence identity are not considered. Alignments performed for the purpose of determining the percentage of amino acid sequence identity can be performed in a variety of known ways, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for the alignment sequences can be determined, including the algorithm required to achieve maximum alignment across the full length of the sequences to be compared.

[0141] As used herein, the terms "immunoglobulin heavy chain constant region" and "Fc region" are used interchangeably and should be understood to refer to the carboxyl-terminal portion of the immunoglobulin heavy chain constant region, or its analogues or portions capable of binding Fc receptors. Each immunoglobulin heavy chain constant region contains four or five domains. These domains are named sequentially as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM without a hinge region. The immunoglobulin heavy chain constant region suitable for use in this invention preferably includes an immunoglobulin hinge region, and preferably also includes a CH3 domain. Most preferably, the immunoglobulin heavy chain constant region includes an immunoglobulin hinge region, a CH2 domain, and a CH3 domain.

[0142] As used herein, the term immunoglobulin “hinge region” should be understood to mean the entire immunoglobulin hinge region, or at least a portion of an immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.

[0143] As used herein, the term "vector" should be understood to mean any nucleic acid containing a nucleotide sequence that can be incorporated into a host cell and recombine with and integrate into the host cell genome, or replicate autonomously as an episome. Such vectors include linear nucleic acids, plasmids, phage particles, granules, RNA vectors, viral vectors, etc. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses.

[0144] As used herein, the terms “gene expression” or “expression” for proteins or peptides should be understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a protein or peptide product. In some embodiments, the expression process may also include, or be followed by, purification; for example, purification may be performed using protein A affinity chromatography or other methods, such as size exclusion chromatography.

[0145] The term "linker," relative to the amino acid linker in a polypeptide, can refer to short peptides such as dimers of two amino acids, trimers of three amino acids, tetramers of four amino acids, pentamers of five amino acids, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235) and (GGGGX). λ (SEQ ID NO:236))n, where X λ It is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, such that the length of the amino acid linker is 25 amino acids or less. Other examples include (X λ GGGG (SEQ ID NO:317) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ GGG (SEQ ID NO:318) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ GG (SEQ ID NO:319) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGGX) λ G (SEQ ID NO:320)) n , where X λIt is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5. Other examples include (X... λ GGG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ GG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGGX) λ )n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5. Other examples include (X... λ GG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ )n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations. Other examples include (X... λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ )n, where X λ The amino acid linker is Q, A, E, or S, and n = 1 to 5, or in some embodiments, n is an integer greater than 5. As a further example, the short polypeptide is a flexible linker or amino acid chain of 1, 2, 3, or 4 amino acids, or 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 a variant of the IL-18 propeptide. In some embodiments, the amino acid linker is a fragment of the IL-18 propeptide or a variant of the IL-18 propeptide; for example, a length of about 30 to 36 amino acids, or a length of about 5 to 10, 11 to 20, 21 to 30, or 31 to 40 amino acids.

[0146] As used herein, in the context of interactions, the term "cis" refers to an interaction between two molecules on the same cell, such as proteins expressed on the same cell. A non-limiting example is the interaction between B7-1 and PD-L1. As used herein, "cis" interactions also include multispecific proteins that simultaneously bind two proteins on the same cell.

[0147] As used herein, in the context of interactions, the term "trans" refers to an interaction between two molecules (such as proteins on different cells) on different cells. A non-limiting example is the interaction between the T cell receptor (TCR) and its ligand (MHC-I / peptide complex), which is the prototype interaction. As used herein, "trans" interactions also include multispecific proteins that bind to one protein on one cell and a second protein on another cell; thus, a "bridge" would be considered to occur trans. "Trans" interactions also include interactions between soluble proteins and cell surface proteins, although these do not form cell bridges.

[0148] As used herein, unless specifically indicated to include only wild-type IL-18 or only IL-18 variants, "IL-18 fusion protein" means a fusion protein that includes wild-type IL-18 or IL-18 variants. Therefore, in a particular embodiment, "IL-18 fusion protein" includes only one of the IL-18 variants as described herein.

[0149] Unless otherwise stated, “antibody” refers to all isotypes (IgG, IgA, IgE, IgM, IgD, and IgY) of immunoglobulins in various monomeric, polymeric, and chimeric forms. The term “antibody” specifically covers VHH antibodies or nanobodies composed of antigen-binding fragments of heavy-chain-only antibodies fused to the Fc group, wherein the VHH is preferably derived from camel-like animals, polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like peptides such as chimeric and humanized antibodies.

[0150] An "antigen-binding fragment" or "bispecific antigen-binding fragment" is any proteinaceous structure that can exhibit binding affinity for a specific antigen. Antigen-binding fragments include those provided by any known technique such as enzyme cleavage, peptide synthesis, and recombinant technology. Some antigen-binding fragments consist of a portion of the intact antibody that retains the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment may contain at least one variable region (heavy chain variable region or light chain variable region) or one or more CDRs of an antibody known to bind a specific antigen. Examples of suitable antigen-binding fragments include, but are not limited to, biantibodies, Fab, F(ab')2, Fc, Fac, and Fv molecules; single-chain (Sc) antibodies; a single antibody light chain; a single antibody heavy chain; 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 chain and one light chain; monovalent fragments consisting of VL, VH, CL, and CH1 domains; or monovalent antibodies; bivalent fragments comprising two Fab fragments linked by disulfide bonds in hinge regions; and fragments substantially composed of V... H Domain and C H1 The Fd fragment is composed of domains; it is essentially composed of the V-arm of the antibody. L Domain and V H The Fv segment is composed of structural domains; it is basically composed of V H Domain-combining dAb fragments (e.g., Ward et al., Nature 341, 544-546 (1989)) and also known as domain antibodies (e.g., Holt et al., Trends Biotechnol., Nov 2003; 21(11):484-90); camel VHHs or nanobodies (e.g., Revets et al., Expert Opin Biol Ther., Jan 2005; 5(1):111-24); isolated complementarity-determining regions (CDRs), etc. All antibody isotypes can be used to generate antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody protein frameworks that can be successfully incorporated into polypeptide segments in orientations that confer affinity to a given antigen of interest (e.g., receptor-ligand pairs; e.g., PD1-Fc fusions targeting PDL1 and PDL2). Antigen-binding fragments can be generated by recombinant methods or by enzymatic or chemical cleavage of intact antibodies. The phrase “antibody or antigen-binding fragment thereof” can be used to refer to one or more amino acid segments of an antibody that are incorporated into the phrase.

[0151] "VHH" refers to a single variable domain on the heavy chain. It can also be called a nanobody. A VHH (or nanobody) contains three CDR domains that constitute most of the complementary site or antigen-binding fragment of a heavy-chain-only antibody (HcAb). Typically, HcAbs are naturally produced by camels and sharks.

[0152] "Single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of immunoglobulins. H ) and light chain variable region (V L A fusion protein that is linked to a short, flexible linker peptide (typically about 10 to 25 amino acids in length).

[0153] In pharmacokinetic studies, half-life (t1 / 2) typically refers to the time required for a 50% decrease in drug concentration. "T1 / 2α" refers to the half-life of the distribution phase, while "T1 / 2β" refers to the half-life of the elimination phase. As some descriptions suggest, the α half-life is the rate of decrease in plasma drug concentration due to the redistribution of the drug from the central compartment to peripheral compartments, while the β half-life is the rate of decrease in plasma drug concentration due to the elimination process caused by metabolism. During the distribution phase, changes in plasma drug concentration primarily reflect the transfer of drug from the circulatory system to internal compartments, rather than loss from the body. However, when the drug reaches equilibrium in plasma and tissues, the decrease in plasma drug concentration is driven by the elimination of the drug from the body; this is known as the elimination phase (late stage).

[0154] "ROR1" (receptor tyrosine kinase-like orphan receptor 1) refers to a member of the receptor tyrosine kinase family with a molecular weight of 106 kDa, which has UniProt accession numbers Q01973 (human) and Q9Z139 (mouse).

[0155] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex consists of six distinct polypeptide chains. These chains include a CD3γ chain (e.g., SwissProt P09693), a CD3δ chain (e.g., SwissProt P04234), two CD3ε chains (e.g., SwissProt P07766), and a CD3ζ chain homodimer (e.g., SwissProt20963), which associate with the α and β chains of the T cell receptor. CD3 further aggregates with the T cell receptor (TCR) or the TCR / CD3 complex to form clusters, which represent key activating receptors expressed on T cells. Thus, cross-linking of CD3 or the TCR produces similar signaling pathways and activates T cells. Unless otherwise stated, the term "CD3" includes any CD3 variant, isotype, and species homolog that is naturally expressed by cells (including T cells) or that can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

[0156] When used in the context of an antibody or antibody fragment, "immunospecific" means binding to one or more epitopes of the protein of interest via a domain encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene, without preferentially binding to other molecules in a sample containing a mixed population of molecules. Phrases such as "anti-[antigen] antibody" (e.g., anti-ROR1 antibody) or "[antigen]-specific antibody" (e.g., "ROR1-specific antibody") are intended to express that the antibody specifically binds to the antigen.

[0157] "Isolation" means that a biological component (such as an antibody) has been substantially separated, prepared separately, or purified from other biological components of the organism in which it is naturally present (i.e., other chromosomes and extrachromosomal DNA and RNA, as well as proteins). Therefore, "isolated" antibodies include antibodies purified using standard purification methods. An "isolated antibody" can be part of a composition, and can still be isolated even if such a composition is not part of the natural environment in which the antibody is naturally present. The term also covers antibodies prepared through recombinant expression in host cells, as well as chemically synthesized antibodies.

[0158] Fusion protein Various embodiments provide a fusion protein comprising a scaffold polypeptide and at least two of the following: (a) one or more of interleukin-15 (IL-15) variants; (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more of receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH. In various embodiments, (b) is one or more IL-18 variants.

[0159] Various embodiments provide a fusion protein comprising a scaffold polypeptide and at least two of the following: (a) one or more variants of interleukin-15 (IL-15); (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more receptor-binding polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding tumor-associated antigens (TAAs). In various embodiments, (b) is one or more IL-18 variants. Polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding TAAs, include those polypeptides described herein and those polypeptides known in the art.

[0160] Various embodiments provide a fusion protein comprising a scaffold polypeptide and at least two of the following: (a) one or more variants of interleukin-15 (IL-15); (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more immune checkpoint targeting fragments. In various embodiments, (b) is one or more IL-18 variants.

[0161] In various embodiments, one or more peptides capable of binding to a TAA include receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH. In various embodiments, the TAA includes EGFR, HER2, or DLL. Peptides capable of binding to a TAA include those described herein and those known in the art.

[0162] In various embodiments, the activating receptors include differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, or the co-stimulatory receptors include differentiation cluster (CD)137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40. Therefore, peptides capable of binding to these activating or co-stimulatory receptors include those peptides described herein and those known in the art.

[0163] In various embodiments, immune checkpoints include, but are not limited to, PD-1, PD-L1, CTLA-4, and LAG-3. In various embodiments, one or more immune checkpoint targeting fragments include immune checkpoint targeting fragments of known antibodies. Examples of anti-PD1 fragments include fragments from pembrolizumab, nivolumab, pildilizumab, AMP-224, AMP-514, spartazumab, cimiprizumab, penamprizumab (AK105), palolizumab (BCD-100), ebbenlimumab (BI754091), toripalimab (JS001), lipusubibumab (LZM009), rivalimab (MGA012), Sym021, dostalimumab (TSR-042), terpolizumab (MGD013), candunizumab (AK104), vodalizumab (XmAb20717), tislelizumab, or PF-06801591 (e.g., Fab, Fv). Other examples of anti-PD1 fragments include fragments (e.g., Fab, Fv) from vopalimumab, camrelizumab, sintilimab, AMP-224, AMP-514, and acrixolimab. Examples of anti-PD-L1 antibodies include, but are not limited to, gorevolimab (BGB-A333), cochilimumab (CK-301), FAZ053, envorimab (KN035), MDX-1105, betifisolimab (MSB2311), adebenone (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824. Thus, for example, fusion proteins may contain, for instance, anti-PD1 fragments (e.g., Fab, Fv) from anti-PD1 antibodies, IL-18 variants and IL-15 variants, and optional linkers.

[0164] In various embodiments, the fusion protein comprises one or more of a scaffold polypeptide and (a) an interleukin-15 (IL-15) variant and (b) an interleukin-18 (IL-18), a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant. In various embodiments, (b) is one or more IL-18 variants.

[0165] In various embodiments, the fusion protein comprises a scaffold polypeptide and all three of the following: (a) one or more of interleukin-15 (IL-15) variants; (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more of receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH. In various embodiments, (b) is one or more IL-18 variants.

[0166] In various embodiments, the fusion protein comprises a scaffold polypeptide and all three of the following: (a) one or more variants of interleukin-15 (IL-15); (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more receptor-binding polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs). In various embodiments, (b) is one or more IL-18 variants.

[0167] In various embodiments, the fusion protein comprises a scaffold polypeptide and all three of the following: (a) one or more of interleukin-15 (IL-15) variants; (b) one or more of interleukin-18 (IL-18), a fragment of that IL-18, an IL-18 variant, or a fragment of that IL-18 variant; and (c) one or more immune checkpoint targeting fragments. In various embodiments, (b) is one or more IL-18 variants.

[0168] In various embodiments, one or more of the IL-15 variants are 2, 3, 4, 5, or 6, or up to 8 IL-15 variants. In various embodiments, one or more of interleukin-18 (IL-18), fragments of IL-18, IL-18 variants, or fragments of IL-18 variants are 2, 3, 4, 5, or 6, or up to 8. In various embodiments, one or more of receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH are 2, 3, 4, 5, or 6, or up to 8.

[0169] In various embodiments, the fusion protein further comprises a receptor-binding polypeptide capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on an immune cell, wherein optionally, the activating receptor includes differentiation cluster (CD)3, CD16, NKp46, or NKG2D, and wherein optionally, the co-stimulatory receptor includes differentiation cluster (CD)137, CD28, DNAM-1, NKp30, CD2, ICOS, OX40, CD40L, and CD40.

[0170] In various embodiments, the fusion protein further comprises a linker located between the scaffold polypeptide and one or more of: (a) one or more of interleukin-15 (IL-15) variants; (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; or (c) one or more of receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH.

[0171] In various embodiments, the fusion protein further comprises a linker between the scaffold polypeptide and: (a) one or more variants of interleukin-15 (IL-15); (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more receptor-binding polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs). In various embodiments, (b) is one or more IL-18 variants.

[0172] In various embodiments, the fusion protein further comprises a linker located between the scaffold polypeptide and: (a) one or more variants of interleukin-15 (IL-15); (b) one or more of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (c) one or more immune checkpoint targeting fragments. In various embodiments, (b) is one or more IL-18 variants.

[0173] In various embodiments, the IL-15 variant, interleukin 18 (IL-18), a fragment of the IL-18, an IL-18 variant or a fragment of the IL-18 variant or receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH are each independently fused to the C-terminus of the scaffold peptide.

[0174] In various embodiments, IL-15 variants, interleukin 18 (IL-18), fragments of IL-18, IL-18 variants or fragments of IL-18 variants or receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH are each independently fused to the N-terminus of the scaffold peptide.

[0175] In various embodiments, an IL-15 variant, IL-18, a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant, and a receptor-binding polypeptide capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs), are each independently fused to the C-terminus of the scaffold polypeptide. In various embodiments, (b) is one or more IL-18 variants.

[0176] In various embodiments, an IL-15 variant, IL-18, a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant, and a receptor-binding polypeptide capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs), are each independently fused to the N-terminus of the scaffold polypeptide. In various embodiments, (b) is one or more IL-18 variants.

[0177] In various embodiments, an IL-15 variant, IL-18, a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant and an immune checkpoint targeting fragment are each independently fused to the C-terminus of the scaffold peptide. In various embodiments, (b) is one or more IL-18 variants.

[0178] In various embodiments, an IL-15 variant, IL-18, a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant and an immune checkpoint targeting fragment are each independently fused to the N-terminus of the scaffold peptide. In various embodiments, (b) is one or more IL-18 variants.

[0179] In various embodiments, the scaffold peptide is an antibody, and one or more peptides, including IL-15 variants, IL-18, fragments of IL-18, IL-18 variants, or fragments of IL-18 variants, as well as receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or capable of binding to tumor-associated antigens (TAAs), are each independently fused to either the C-terminus or N-terminus of the heavy or light chain, or to the C-terminus of the antibody. H 2. Merge either the structural domain or the hinge region.

[0180] In various embodiments, the scaffold peptide is an antibody, and an IL-15 variant, interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or a receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH are each independently fused to either the C-terminus or N-terminus of the heavy or light chain, or to the C-terminus of the antibody. H 2. Merge either the structural domain or the hinge region.

[0181] In various embodiments, the scaffold peptide is an antibody, and an IL-15 variant, interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant or an immune checkpoint targeting fragment are each independently fused to either the C-terminus or N-terminus of the heavy or light chain, or to the C-terminus of the antibody. H 2. Merge either the structural domain or the hinge region.

[0182] In various embodiments, the scaffold polypeptide is an Fc region, and one or more polypeptides, including an IL-15 variant, interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant or a fragment of the IL-18 variant, or a receptor-binding polypeptide capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on an immune cell, or a tumor-associated antigen (TAA), are each independently fused to either the C-terminus or the N-terminus of the Fc region.

[0183] In various embodiments, the scaffold peptide is an Fc region, and an IL-15 variant, interleukin 18 (IL-18), a fragment of the IL-18, an IL-18 variant or a fragment of the IL-18 variant or a receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH are each independently fused to either the C-terminus or the N-terminus of the Fc region.

[0184] In various embodiments, the scaffold peptide is an Fc region, and an IL-15 variant, interleukin-18 (IL-18), a fragment of the IL-18, an IL-18 variant, or a fragment of the IL-18 variant or an immune checkpoint targeting fragment are each independently fused to either the C-terminus or the N-terminus of the Fc region.

[0185] 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. Other examples of antibodies include, but are not limited 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 antibody can be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibody is a human antibody or a humanized antibody.

[0186] In various implementations, the antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, pildizumab, AMP-224, AMP-514, spartazumab, cimiprizumab, penampilimab (AK105), palolizumab (BCD-100), ebbenlimumab (BI 754091), toripalimab (JS001), lipusubibumab (LZM009), rivalimab (MGA012), Sym021, dostalimumab (TSR-042), terpolilimumab (MGD013), candunilimumab (AK104), vordalilimumab (XmAb20717), tislelizumab, PF-06801591, and pluripotent cytotoxic T lymphocytes expressing anti-PD1 antibodies (PIK-PD-1). Examples of anti-PD-L1 antibodies include, but are not limited to, gliovelimab (BGB-A333), cosivelimab (CK-301), FAZ053, envorimab (KN035), MDX-1105, betifisolimab (MSB2311), adebenone (SHR-1316), atezolizumab, averumab, durvalumab, BMS-936559, CK-301, and M7824.

[0187] In various embodiments, the two arms (or chains) of the constant region of the immunoglobulin heavy chain (e.g., an Fc polypeptide) can undergo heterodimerization by generating a "mortar and pestle" (KiH) mutation in the CH3 domain. This structural feature in the polypeptide arms allows the assembly of two half-antibodies (e.g., an Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each containing a CH3 domain, wherein the two polypeptides interlock at an engineered interface within the CH3 domain, and one polypeptide contains an engineered protrusion ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume larger than the original residue; and the other polypeptide contains an engineered cavity ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume smaller than the original residue. In some embodiments, the engineered interface of the heteromultimer comprises at least two pairs of protrusion-cavity mutants. The volume and accessible surface area of ​​each amino acid are described in AA Zamyatnin, Prog. Biophys., Mol. Biol. 24: 107-123, 1972; and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, the lead-in residues used to form the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the lead-in residues used to form the cavity may be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residues used to form the cavity have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "groove" / protrusion chain and the T366S / L368A / Y407V mutation in the CH3 domain of the "mortar" / cavity chain. Additionally, the KiH conformation can be coupled with further mutations to allow SS disulfide bond linkage between the two chains. In this conformation, the protein / peptide is a heterodimer of the KiH conformation, with IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) linked to one and only one of two (or more) immunoglobulin heavy chain constant regions / chains (i.e., groove or mortar).

[0188] In various embodiments, the scaffold polypeptide is a crystallizable fragment (Fc) region or a fragment thereof. In various embodiments, the crystallizable fragment (Fc) region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1. In various embodiments, the crystallizable fragment (Fc) region is KiH (KiH) Fc.

[0189] In various embodiments, the Fc region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1. In various embodiments, the Fc region can be human IgG1, IgG2, or IgG4.

[0190] In some embodiments, two or more arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) may contain designs such as another symmetric-asymmetric stereocomplementary design (e.g., HA-TF, ZW1), charge-exchange interaction (DD-KK), charge-stereocomplementary exchange plus additional long-range electrostatic interaction (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic redirection, A107, Duobody, thereby forming a heterodimer / heteromultimer. Further descriptions of these conformations and exemplary mutations / residues can be found in FrontImmunol. 2016; 7: 394.

[0191] In various embodiments, the scaffold peptide is selected from crystallizable fragment (Fc) regions, human serum albumin (HSA), β2 microglobulin, transferrin, fragment antigen-binding regions (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, and their fragments.

[0192] In various implementations, the scaffold polypeptide is a polypeptide or protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER).

[0193] Further details and implementation schemes of the scaffold peptides are described herein and are intended to be included as implementation schemes of these multi-cytokine fusion proteins.

[0194] In various implementations, the IL-15 variant contains the sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKE FLQSX 103 VHIVX 108 MFIX 112 TS, in which X1 can be any amino acid. X4 can be any amino acid. X 22 It can be any amino acid. X 30 It can be any amino acid. X 41 It can be any amino acid. X 45 It can be any amino acid. X 49 It is any amino acid except V. X 53 It is any amino acid except E. X 64 It can be any amino acid. X 67 It can be any amino acid. X 68 It can be any amino acid. X 79 It can be any amino acid. X 93 It can be any amino acid. X 103 It can be any amino acid. X 108 It is any amino acid, and X 112 It is any amino acid; (SEQ ID NO:533).

[0195] Further details and implementation schemes of the IL-15 variant component of the fusion protein are described herein and are intended to be included as implementation schemes of these multi-cytokine fusion proteins.

[0196] In various embodiments, the IL-18 variant has an amino acid sequence comprising or composed of amino acid positions 37-193 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250), having 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 are 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 variants contain no more than five amino acid substitutions, except for substituted cysteine. In various embodiments, the IL-18 variants have an amino acid sequence comprising or consisting of amino acid positions 37-193 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251), having one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251.

[0197] In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251), with one or more amino acid substitutions at positions C74, C104, C112, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149.

[0198] In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 are each independently substituted with valine, alanine, or serine. In various embodiments, the one to five amino acid substitutions are one or more of the following: 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.

[0199] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 6B. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 6B further comprises a propeptide having an amino acid sequence selected from the propeptide column in Table 6B, and optionally from the same row as the IL-18 variant. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 6B and a 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 the propeptide. As a specific example, IEQD (SEQ ID NO: 88) may be used.

[0200] In various embodiments, IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant further comprises its propeptide (PP) or a PP variant. In various embodiments, the PP or PP variant may be located at the N-terminus relative to IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0201] In various embodiments, the fusion protein further includes one or more cleavage sites, and the fusion protein can be cleaved at one or more cleavage sites by one or more proteases.

[0202] In various embodiments, the one or more cleavage sites are located between IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant and the scaffold polypeptide; or within PP, between PP or a PP variant and IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; or within PP, between PP or a PP variant and the scaffold polypeptide; or within IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; or within PP; or combinations thereof.

[0203] Further details and embodiments of IL-18 of the fusion protein, fragments of the IL-18, variants of the IL-18, or fragment components of the variants of the IL-18 are described herein and are intended to be included as embodiments of these multi-cytokine fusion proteins.

[0204] In various embodiments, 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 (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 substitutions of amino acid residues 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 substitutions of amino acid residues in the polypeptide having SEQ ID NO:327, wherein the variant of the polypeptide having SEQ ID NO:325 comprises one or more deletions, additions, or substitutions of amino acid residues 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 substitutions of amino acid residues in the polypeptide having SEQ ID NO:327, wherein the variant of the polypeptide having SEQ ID NO:325 ... Variants of the polypeptide with SEQ ID NO:327 include one or more deletions, additions, or substitutions of amino acid residues in the polypeptide with SEQ ID NO:327, and wherein: the variants of the polypeptide with SEQ ID NO:325 and the variants of the polypeptide with SEQ ID NO:327 do not replace cysteine ​​residues in the polypeptide with SEQ ID NO:325 and the polypeptide with SEQ ID NO:327, or the variants of the polypeptide with SEQ ID NO:325 and the variants of the polypeptide with SEQ ID NO:327 replace one or two cysteine ​​residues in the polypeptide with SEQ ID NO:325 and / or one or two cysteine ​​residues in the polypeptide with SEQ ID NO:327 with amino acids containing cross-linking functional groups.

[0205] Further details and implementation schemes of the ROR1 VHH component of the fusion protein are described herein and are intended to be included as implementation schemes of these multi-cytokine fusion proteins.

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

[0207] In embodiments in which the fusion protein comprises a scaffold polypeptide and one or more of (a) interleukin-15 (IL-15) variants and one or more of (b) IL-18 variants, the (a) interleukin-15 (IL-15) variants and the (b) IL-18 variants may be included as described herein and are intended to be included as embodiments of these multi-cytokine fusion proteins.

[0208] In various embodiments, a fusion protein comprising one or more of the following: a) interleukin-15 (IL-15) variants and (b) interleukin-18 (IL-18), a fragment of the IL-18, an IL-18 variant, or a fragment of the 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.

[0209] In various embodiments, the fusion protein comprising the scaffold polypeptide and one or more of (a) interleukin-15 (IL-15) variants and (b) interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an 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.

[0210] Polynucleotides, expression vectors, cells, and methods for producing fusion proteins .

[0211] Various implementations provide a polynucleotide that encodes any of the fusion proteins of the present invention as described herein.

[0212] Various implementations provide an expression vector comprising any of the polynucleotides of the present invention as described herein.

[0213] Various embodiments provide a cell transfected with any of the expression vectors 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 (mouse myeloma cell line), and PER.C6. ® Cells and human embryonic kidney cells (HEK cells). In various embodiments, the cell is a bacterial cell or a yeast cell.

[0214] Various embodiments provide a method for producing any of the fusion proteins of the present invention described herein, the method comprising: culturing any of the cells of the present invention described herein in a cell culture medium to allow the production of the fusion protein, and optionally secreting it into the cell culture medium. In various embodiments, the method further includes isolating the fusion protein. In various embodiments, the method further includes purifying the fusion protein.

[0215] In some implementations, the fusion protein is generated using a Chinese hamster ovary (CHO) expression system by a method comprising the following steps: (1) cell resuscitation, in which frozen CHO cells can be resuscitated in a water bath at 37°C; and (2) cell passage culture, in which the cells can be passaged to adjust the cell density to 6 × 10⁻⁶. 6 (3) Transfection and expression: use solution 1 (where the plasmid is diluted with diluent) and solution 2 (where the transfection reagent is diluted with diluent), then mix solution 1, solution 2 and CHO cells, and then incubate the mixture in a shaker at 32°C for 12 to 14 days for expression, and collect the culture supernatant after centrifugation.

[0216] In some embodiments, a purification process is performed after the fusion protein is expressed. In some embodiments, the purification method includes the following steps: (1) washing the column with binding buffer (10 volumes) at a flow rate of 1 mL / min; (2) loading a sample containing the fusion protein at a flow rate of 1 mL / min; (3) washing the column with 10 volumes of PBS buffer at a flow rate of 1 mL / min; (4) eluting the protein from the column with 40 mM sodium citrate (pH 3.4); optionally, the eluted sample may be collected in a tube (1 mL / min) and the optical density (OD) may be measured at 280 nm using NanoDrop; and (5) performing dialysis, for example, overnight dialysis with PBS buffer in a dialysis bag.

[0217] How to use Various embodiments provide a method for activating and promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, the method comprising administering to a subject in need any of the fusion proteins of the present invention described herein. The expansion of T cells, B cells, or natural killer (NK) cells does not require binding to tumor-associated antigens, such as ROR1. In various embodiments, the subject has cancer.

[0218] Various embodiments provide a method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject any of the fusion proteins of the present invention described herein. In various embodiments, the disease or symptom is cancer.

[0219] Various embodiments provide a method for activating and promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, the method comprising administering to a subject in need at least two of the following: (a) an interleukin-15 (IL-15) variant or IL-15 fusion protein; (b) interleukin-18 (IL-18), a fragment of the IL-18, an 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 (ROR1) VHH and a receptor-binding polypeptide capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on an immune cell, wherein optionally, the activating receptor comprises differentiation cluster (CD)3, CD16, NKp46, or NKG2D, and wherein optionally, the co-stimulatory receptor comprises differentiation cluster (CD)137, CD28, DNAM-1, NKp30, CD2, ICOS, OX40, CD40L, and CD40. In various implementation schemes, the subject has cancer. The expansion of T cells, B cells, or natural killer (NK) cells does not require binding to tumor-associated antigens such as ROR1.

[0220] Various implementations provide a method for activating, promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, the method comprising administering to a subject in need at least two of the following: (a) an interleukin-15 (IL-15) variant or IL-15 fusion protein; (b) interleukin-18 (IL-18), a fragment of IL-18, an 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 (ROR1) VHH and a binding site on... The active receptor-binding polypeptide of an activating receptor and / or a co-stimulatory receptor expressed on immune cells, wherein optionally, the activating receptor includes differentiation cluster (CD)3, CD16, NKp46, or NKG2D, and wherein optionally, the co-stimulatory receptor includes differentiation cluster (CD)137, CD28, DNAM-1, NKp30, CD2, ICOS, OX40, CD40L, and CD40; and (d) one or more receptor-binding polypeptides capable of binding to the activating receptor and / or co-stimulatory receptor expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs). In various embodiments, the subject has cancer. Expansion of T cells, B cells, or natural killer (NK) cells does not require binding to tumor-associated antigens, such as ROR1.

[0221] A method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject in need at least two of the following: (a) an interleukin-15 (IL-15) variant or IL-15 fusion protein; (b) interleukin-18 (IL-18), a fragment of the IL-18, an 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 (ROR1) VHH and a receptor-binding polypeptide capable of binding to an activated receptor and / or a co-stimulatory receptor expressed on an immune cell, wherein optionally, the activated receptor comprises differentiation cluster (CD)3, CD16, NKp46, or NKG2D, and wherein optionally, the co-stimulatory receptor comprises differentiation cluster (CD)137, CD28, DNAM-1, NKp30, CD2, ICOS, OX40, CD40L, and CD40. In various implementation schemes, the disease or symptom is cancer.

[0222] A method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising administering to the subject in need at least two of the following: (a) an interleukin-15 (IL-15) variant or IL-15 fusion protein; (b) interleukin-18 (IL-18), a fragment of the IL-18, an 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 (ROR1) VHH and an expression capable of binding to immune cells. The receptor-binding polypeptide of an activating receptor and / or a co-stimulatory receptor, wherein optionally, the activating receptor includes differentiation cluster (CD)3, CD16, NKp46, or NKG2D, and wherein optionally, the co-stimulatory receptor includes differentiation cluster (CD)137, CD28, DNAM-1, NKp30, CD2, ICOS, OX40, CD40L, and CD40; and (d) one or more receptor-binding polypeptides capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on immune cells, or one or more polypeptides capable of binding to a tumor-associated antigen (TAA). In various embodiments, the disease or symptom is cancer.

[0223] In some implementations, the cancer is a ROR1-expressing cancer, such as lung cancer, hematologic malignancies, breast cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cell carcinoma, uterine cancer, bladder cancer, kidney cancer, melanoma, thyroid cancer, myeloid leukemia, mantle cell lymphoma, or multiple myeloma. ROR1-expressing cancers can be lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). ROR1-expressing cancers can be hematologic malignancies, such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS, low-risk or high-risk), acute lymphoblastic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell tumor (DPDCN). ROR1-expressing cancers can be breast cancer. ROR1-expressing cancers can be prostate cancer. ROR1-expressing cancers can be pancreatic cancer. ROR1-expressing cancers can be colon cancer. Cancers expressing ROR1 can include ovarian cancer, renal cell carcinoma, uterine cancer, and melanoma.

[0224] IL-15 variant details and implementation scheme This document describes IL-15 variants that can be used in the multi-cytokine fusion protein of the present invention. Various embodiments provide an interleukin-15 (IL-15) variant comprising the sequence of Formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKE FLQSX 103 VHIVX 108 MFIX 112 TS, in which X1 can be any amino acid. X4 can be any amino acid. X 22 It can be any amino acid. X 30 It can be any amino acid. X 41 It can be any amino acid. X 45 It can be any amino acid. X 49 It is any amino acid except V. X 53 It is any amino acid except E. X 64 It can be any amino acid. X 67 It can be any amino acid. X 68 It can be any amino acid. X 79 It can be any amino acid. X 93 It can be any amino acid. X 103 It can be any amino acid. X 108 It is any amino acid, and X 112 It is any amino acid; (SEQ ID NO:533).

[0225] In various embodiments of the IL-15 variant, X1 is N (SEQ ID NO:534), X4 is N (SEQ ID NO:535), X 22 It is D (SEQ ID NO:536), X 30 It is D (SEQ ID NO:537), X41 It is K (SEQ ID NO:538), X 45 It is L (SEQ ID NO: 539), X 64 It is E (SEQ ID NO:540), X 67 It is I (SEQ ID NO:541), X 68 It is I (SEQ ID NO:542), X 79 It is N (SEQ ID NO:543), X 93 It is E (SEQ ID NO:544), X 103 It is F (SEQ ID NO:545), X 108 It is Q (SEQ ID NO:546), or X 112 It is N (SEQ ID NO: 547), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of them.

[0226] In various embodiments of the IL-15 variant, X1 is N (SEQ ID NO:534), X4 is N (SEQ ID NO:535), X 22 It is D (SEQ ID NO:536), X 30 It is D (SEQ ID NO:537), X 41 It is K (SEQ ID NO:538), X 45 It is L (SEQ ID NO: 539), X 49 It is R or K (SEQ ID NO:548), X 53 It is G, K, I or A (SEQ ID NO:549), X 64 It is E (SEQ ID NO:540), X 67 It is I (SEQ ID NO:541), X 68 It is I (SEQ ID NO:542), X 79 It is N (SEQ ID NO:543), X 93 It is E (SEQ ID NO:544), X 103 It is F (SEQ ID NO:545), X 108 It is Q (SEQ ID NO:546), or X 112It is N (SEQ ID NO: 547), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of them.

[0227] In various embodiments of the IL-15 variant, X1 is N or G (SEQ ID NO:550), X4 is N, K, or L (SEQ ID NO:551), X 22 It is D or A (SEQ ID NO:552), X 30 It is D or N (SEQ ID NO:553), X 41 It is K or Q (SEQ ID NO:554), X 45 It is L or S (SEQ ID NO:555), X 49 It is R or K (SEQ ID NO:548), X 53 It is G, K, I or A (SEQ ID NO:549), X 64 It is E or Q (SEQ ID NO:556), X 67 It is I or T (SEQ ID NO:557), X 68 It is I or S (SEQ ID NO:558), X 79 It is N or Y (SEQ ID NO:559), X 93 It is E or A (SEQ ID NO:560), X 103 It is F or L (SEQ ID NO:561), X 108 It is Q or T (SEQ ID NO:562), or X 112 It is N or R (SEQ ID NO: 563), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of them.

[0228] In various implementations of IL-15 variants, X 49 It is R or K, and X 53 It is G, K, I, or A; (SEQ ID NO:564). In various embodiments of the IL-15 variant, X 30 It is N (SEQ ID NO:565). In various embodiments of the IL-15 variant, X 64X1 is Q (SEQ ID NO:566). In various embodiments of the IL-15 variant, X1 is G (SEQ ID NO:567).

[0229] In various embodiments, the IL-15 variants are selected from those in Table 1A. In various embodiments, IL-15 is selected from those in Table 3A.

[0230] In various embodiments, the IL-15 variant is not the IL-15 variant described in U.S. Patent Application Publication No. US 2019 / 0263877, which is incorporated herein by reference.

[0231] This document also describes fusion proteins comprising an IL-15 variant and a scaffold peptide, which can be used in various methods associated with the administration of the individual fusion proteins described herein. Various embodiments of the invention provide a fusion protein comprising an IL-15 variant as described herein and a scaffold peptide. These IL-15 fusion proteins can be used according to the methods of the invention as described herein.

[0232] In various embodiments, the fusion protein comprises two or more IL-15 variants as described herein; and a scaffold peptide. In various embodiments, the two or more IL-15 variants may be three, four, five, or six IL-15 variants. In various embodiments, the two or more IL-15 variants may be up to eight IL-15 variants.

[0233] In various embodiments, the IL-15 variant is fused to the C-terminus of the scaffold peptide. In other embodiments, the IL-15 variant is fused to the N-terminus of the scaffold peptide.

[0234] In some other embodiments, the scaffold peptide is an antibody, and the IL-15 variant is fused to either the C-terminus or N-terminus of the heavy or light chain, or to C... H 2. Merge either the structural domain or the hinge region (e.g., located in C) H 2. Structural Domains and C H 1. Between structural domains.

[0235] 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.

[0236] In various implementations, the antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, pildizumab, AMP-224, AMP-514, spartazumab, cimiprizumab, penampilimab (AK105), palolizumab (BCD-100), ebbenlimumab (BI 754091), toripalimab (JS001), lipusubibumab (LZM009), rivalimab (MGA012), Sym021, dostalimumab (TSR-042), terpolilimumab (MGD013), candunilimumab (AK104), vordalilimumab (XmAb20717), tislelizumab, PF-06801591, and pluripotent cytotoxic T lymphocytes expressing anti-PD1 antibodies (PIK-PD-1). Examples of anti-PD-L1 antibodies include, but are not limited to, gliovelimab (BGB-A333), cosivelimab (CK-301), FAZ053, envorimab (KN035), MDX-1105, betifisolimab (MSB2311), adebenone (SHR-1316), atezolizumab, averumab, durvalumab, BMS-936559, CK-301, and M7824.

[0237] In various embodiments, the scaffold peptide is an Fc region or a fragment thereof. In various embodiments, the scaffold peptide is an Fc region or a fragment thereof, and the scaffold peptide does not contain Fab.

[0238] In various embodiments, the two arms (or chains) of the constant region of the immunoglobulin heavy chain (e.g., an Fc polypeptide) can undergo heterodimerization by generating a "mortar and pestle" (KiH) mutation in the CH3 domain. This structural feature in the polypeptide arms allows the assembly of two half-antibodies (e.g., an Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each containing a CH3 domain, wherein the two polypeptides interlock at an engineered interface within the CH3 domain, and one polypeptide contains an engineered protrusion ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume larger than the original residue; and the other polypeptide contains an engineered cavity ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume smaller than the original residue. In some embodiments, the engineered interface of the heteromultimer comprises at least two pairs of protrusion-cavity mutants. The volume and accessible surface area of ​​each amino acid are described in AA Zamyatnin, Prog. Biophys., Mol. Biol. 24: 107-123, 1972; and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, the lead-in residues used to form the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the lead-in residues used to form the cavity may be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residues used to form the cavity have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "groove" / protrusion chain and the T366S / L368A / Y407V mutation in the CH3 domain of the "mortar" / cavity chain. Additionally, the KiH conformation can be coupled with further mutations to allow SS disulfide bond linkage between the two chains. In this conformation, the protein / peptide is a heterodimer of the KiH conformation, with IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) linked to one and only one of two (or more) immunoglobulin heavy chain constant regions / chains (i.e., groove or mortar).

[0239] Therefore, in some implementations, the Fc region is the mortar and pestle (KiH) Fc.

[0240] In various embodiments, the IL-15 variant is fused with the pestle of the KiH Fc. In various embodiments, the IL-15 variant is fused with the mortar of the KiH Fc.

[0241] In various embodiments, the Fc region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1. In various embodiments, the Fc region can be human IgG1, IgG2, or IgG4.

[0242] In some embodiments, two or more arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) may contain designs such as another symmetric-asymmetric stereocomplementary design (e.g., HA-TF, ZW1), charge-exchange interaction (DD-KK), charge-stereocomplementary exchange plus additional long-range electrostatic interaction (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic redirection, A107, Duobody, thereby forming a heterodimer / heteromultimer. Further descriptions of these conformations and exemplary mutations / residues can be found in FrontImmunol. 2016; 7: 394.

[0243] In various embodiments, the scaffold peptide of the fusion protein includes globular proteins, human serum albumin (HSA), β2 microglobulin, transferrin, fragment antigen-binding domains (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, or fragments thereof.

[0244] In various embodiments, one or more peptides may be inserted between an antibody and an IL-15 variant. In various embodiments, the peptide may be inserted into or conjugated at the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the antibody. In various embodiments, the peptide comprises a peptide linker conjugated to the IL-15 variant and the antibody.

[0245] In various embodiments, one or more peptides may be inserted between the IL-15 variant and the Fc region of the IL-15 fusion protein. In various embodiments, the peptide may be inserted or conjugated at the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the Fc region. In various embodiments, the peptide includes a peptide linker conjugating the IL-5 variant and the Fc region. In these embodiments, the fusion protein does not include the Fab region of the antibody.

[0246] Antibodies that can fuse with IL-15 may 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 antibody may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibody is a human antibody or a humanized antibody.

[0247] In various embodiments, the IL-15 fusion protein is the IL-15 fusion protein listed in Table 1A. That is, an IL-15 fusion protein comprising peptide 1, peptide 2, and peptide 3.

[0248] In various embodiments, the IL-15 fusion protein comprises peptides 1, 2, and 3 listed in Table 1A, differing only in that the linker in peptide 2 is a different peptide linker. For example, this linker may be a flexible linker, typically about 10 to 25 amino acids in length. Other examples of such linkers include, but are not limited to, two-amino acid dimers, three-amino acid trimers, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 235), (GGGGX). λ (SEQ ID NO:236))n and (X λ GGGG (SEQ ID NO:317))n, where X λ It is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, so that the length of the amino acid linker is 25 amino acids or less.

[0249] In various implementations, the IL-15 fusion protein includes the IL-15 variants listed in Table 1A or Table 3B.

[0250] Various embodiments provide a polynucleotide that encodes the IL-15 variant of the present invention described herein. For example, the nucleic acid sequence may encode an IL-15 variant or an IL-15 fusion protein in the 5' to 3' orientation.

[0251] Various embodiments provide a polynucleotide that encodes the IL-15 fusion protein of the present invention as described herein. For example, the nucleic acid sequence may encode an IL-15 variant in the 5' to 3' orientation.

[0252] Therefore, the polynucleotides encoding the IL-15 fusion protein of the present invention include polynucleotides 1, 2, and 3 listed in Table 2A. In other embodiments, the polynucleotides encoding the IL-15 fusion protein of the present invention include polynucleotides 1, 2, and 3 listed in Table 2A, except that the nucleotides encoding the adapter encode different adapters.

[0253] For example, the linker can be a flexible linker, typically about 10 to 25 amino acids in length. Other examples of such linkers include, but are not limited to, dimers of two amino acids, trimers of three amino acids, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX). λ (SEQ ID NO:236))n and (X λ GGGG(SEQ ID NO:317))n, where X λIt is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, so that the length of the amino acid linker is 25 amino acids or less. Therefore, the nucleotide encoding the linker in polynucleotide 2 will instead encode one of these aforementioned linkers.

[0254] Exemplary polynucleotides are shown in Table 2A.

[0255] Table 4. Protein expression yield IL-18 Details and Implementation Plan The full text of international application No. PCT / US2023 / 071663, entitled “IL-18 Fusion Proteins and Methods of Producing IL-18”, filed on August 4, 2023, and international application No. PCT / US2024 / 027248, entitled “Methods of Using IL-18 Fusion Proteins”, filed on May 1, 2024, is incorporated herein by reference as if listed in its entirety.

[0256] This document describes IL-18 variants that can be used in the multi-cytokine fusion protein of the present invention. It also describes an IL-18 fusion protein comprising an IL-18 variant, which can be used in methods associated with the administration of a separate fusion protein.

[0257] Various embodiments provide one or more fusion proteins, each comprising (i) IL-18, a fragment of IL-18, a variant of IL-18, or a fragment of a variant of IL-18; and (ii) a first protein, or a fragment of said protein, capable of translocating into the endoplasmic reticulum (ER), the first protein comprising an engineered cytoplasmic or nuclear protein—translocated into the ER by adding a signal peptide / leader sequence to the N-terminus of such engineered protein. Preferably, the protein capable of translocating into the ER has an amino acid sequence that initiates the transport of a protein (e.g., IL-18, a fragment of it, a variant of it, or a fragment of a variant of it) across the membrane of the ER. In various embodiments, the fusion protein also comprises an amino acid linker. For example, the amino acid linker may be between (a) the protein capable of translocating into the ER and (b) a propeptide (or a variant) or IL-18 (or a variant).

[0258] In some embodiments, one or more fusion proteins do not contain IL-18 propeptide or a variant thereof. The terms "IL-18 propeptide," "propeptide," or "PP" are used interchangeably in this invention, describing an amino acid sequence linked to IL-18 or an IL-18 variant precursor in which the removal of this amino acid sequence yields mature IL-18 or a fragment thereof, or an IL-18 variant or a fragment thereof. For example, the IL-18 propeptide may have a sequence of amino acid residues 1 to 36 of Uniprot ID Q14116.

[0259] In some embodiments, one or more fusion proteins further comprise a propeptide (PP) or a variant thereof. Examples of propeptide variants are provided herein, including those in Table 6B. This can inactivate IL-18 or an IL-18 variant, thus the propeptide is directly or indirectly linked to IL-18 or an IL-18 variant to form precursor IL-18 or a precursor IL-18 variant. Preferably, the PP or a variant thereof is N-terminus of the fusion protein relative to IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof). In some embodiments, one or more fusion proteins further comprise a cleavage site, which is preferably based on a peptide substrate sensitive to enzyme / protease cleavage. The cleavage site may be located within the PP, between the PP or a variant thereof (if present) and IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof); or between a protein capable of translocating to the ER and the PP (if present); or between a protein capable of translocating to the ER and a fragment thereof, a variant thereof, or a fragment of IL-18, particularly in the absence of the PP. In some embodiments, when the PP is present, the cleavage site is located within the PP. In a further embodiment, one or more fusion proteins comprise (i) IL-18, a fragment of IL-18, a variant of IL-18, or a fragment of a variant of IL-18; (ii) a propeptide (PP) or a variant thereof that inactivates IL-18; and a cleavage site.

[0260] Examples of propeptide variants include polypeptides having AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, wherein X1 is any amino acid other than cysteine ​​(SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76).

[0261] In various embodiments, the fusion protein does not contain a polypeptide consisting of (i) a propeptide or a propeptide variant and (ii) a mature IL-18 or a mature IL-18 variant, wherein X1 is L or absent, X2 is E or absent, X3 is S or absent, and X4 is D or absent.

[0262] In all aspects of the fusion protein, IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) is linked via a polypeptide bond to a first protein capable of translocating into the ER. The fusion protein may have various conformations. Preferably, the N-terminus of IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) is linked directly or indirectly via a polypeptide bond to the C-terminus of the first protein capable of translocating into the ER.

[0263] In other embodiments, the C-terminus of IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) is directly or indirectly linked via a polypeptide bond to the N-terminus of a first protein capable of translocating in the ER. As a non-limiting example, an IL-18 variant (or IL-18, a fragment thereof, or a fragment thereof) is fused to the N-terminus of the pestle of a pestle-and-mortar heterodimer IgG1 protein having or not having a propeptide (pp).

[0264] In a further embodiment, when the C-terminus of IL-18 is attached to the N-terminus of a first protein capable of translocating in (or across) the ER, a second protein capable of translocating across the ER typically fuses to the N-terminus of IL-18 to mediate masking. It is envisioned that the fusion protein also comprises (iii) a second protein capable of translocating into / across the ER, or a “scaffold,” such as a heat shock protein (HSP) that may not translocate across the ER. In some embodiments, if an HSP (nuclear protein) or cytoplasmic protein fuses to the N-terminus of IL-18 to mediate masking, it typically requires a signal peptide fused to the N-terminus of the “scaffold” to mediate ER transport; and if the scaffold fuses to the C-terminus of IL-18 to stabilize the complex, the second protein capable of translocating across the ER typically fuses to the N-terminus of IL-18 to mediate masking. Therefore, in some embodiments, IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is located at the C-terminus of the fusion protein; in some embodiments, the N-terminus of IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is located at the C-terminus relative to a first protein capable of translocating in the ER, and the C-terminus of IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is located at the N-terminus relative to a second protein capable of translocating in the ER. The “first” or “second” protein capable of translocating in the 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.

[0265] Some embodiments specify that the first protein / peptide capable of translocating to the ER comprises an immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin heavy chain constant region domain selected from the group consisting of: a CH2 domain, a CH3 domain, and a CH4 domain, or a combination thereof. In some embodiments, the immunoglobulin heavy chain constant region comprises both a CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin heavy chain constant region lacks at least a CH1 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 IL-18. In other embodiments, the immunoglobulin heavy chain constant region is an immunoglobulin heavy chain constant region present in the same species as the organism that has been transformed or transfected with a nucleic acid molecule encoding a fusion protein or a fusion protein precursor. Further embodiments specify that the fusion protein lacks an immunoglobulin variable domain (V... H ).

[0266] In various embodiments, IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) is identical in sequence to human-derived IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof), and the immunoglobulin heavy chain constant region comprises a hinge region and a CH2 domain or a CH3 domain, and more preferably comprises both a hinge region and a CH2 domain and a CH3 domain.

[0267] In various embodiments, IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) is at least 95%, 90%, or 85% sequence identical to human-derived IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof), but has amino acid substitutions or other modifications that reduce the affinity of IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) for IL-18BP. It is contemplated that the immunoglobulin heavy chain constant region suitable for use in this invention can be derived from any of the five immunoglobulin classes known in the art as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ). However, immunoglobulin heavy chain constant regions derived from the IgG class are preferred. Furthermore, the immunoglobulin heavy chain constant region can be derived from any IgG antibody subclass known in the art as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain constant region domains exhibit cross-homology among immunoglobulin classes. For example, the CH2 domain of IgG is homologous to the CH2 domains of IgA and IgD, and homologous to the CH3 domains of IgM and IgE. Preferred immunoglobulin heavy chain constant regions include protein domains corresponding to the CH2 and CH3 regions of IgG, or functional portions or derivatives thereof. Further description of immunoglobulin heavy chain constant regions is discussed in detail in U.S. Patent Nos. 5,541,087 and 5,726,044, which are incorporated herein by reference.

[0268] In several embodiments, the protein / peptide to be fused with IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is a dimer of two immunoglobulin heavy chain constant regions / chains, optionally cross-linked by a pair of disulfide bonds between cysteine ​​residues on adjacent hinge regions. In some embodiments, the hinge regions may have an upper hinge domain, a core hinge domain, and a lower hinge domain. In some embodiments, the upper portion of the hinge domain may include or remove cysteine ​​residues known to form disulfide bonds with light chains or fabs, producing sequences such as EPKSC (SEQ ID NO:241), EPKSS (SEQ ID NO:242), or EPKSA (SEQ ID NO:243). For example, fusion proteins comprising IgG1-based ER translocation proteins (except FUSE-501, FUSE-503, and FUSE-509) may have had cysteine ​​removed from the hinge region, such as EPKSS (SEQ ID NO:242) in IgG1-based ER translocation proteins, except for FUSE-507 (FUSE-507 has EPKSA (SEQ ID NO:243) in the hinge region). The hinge region may also contain a core hinge domain, such as containing the sequence CPCCPP (SEQ ID NO:244) or a variant in which cysteine ​​is substituted. The hinge region may also contain a lower hinge domain, such as containing the sequences APELLGGP (SEQ ID NO:245) or APEAAGGP (SEQ ID NO:246). In another example, FUSE-509 has an IgG4-based ER translocation protein that uses the hinge region as described in Chiu et al., Antibodies 2019, 8(4), 55, 2019. Although, as shown in the accompanying figures, constructs containing immunoglobulin hinge regions are preferred, the present invention contemplates the possibility of crosslinking at other locations as needed. Furthermore, in some cases, two or more monomers can nonvalently associate to produce dimers or multimers. Where the protein / peptide is a dimeric aspect of two immunoglobulin heavy chain constant regions / chains, IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) is linked to one and only one of the two (or more) immunoglobulin heavy chain constant regions / chains. In the case of forming homodimers of wild-type IgG-Fc, in various cases, IL-18 is positioned at the C-terminus of each monomer of the Fc, resulting in two IL-18s positioned at the C-terminus of the Fc. In some cases, when a wild-type Fc fused with one IL-18 is mixed with another wild-type Fc not fused with IL-18, heterodimers can be formed (e.g., in purification steps). In other respects, IL-18 (or a fragment thereof, a variant thereof, or a fragment thereof) connects each of the two (or more) immunoglobulin heavy chain constant regions / chains in the fusion protein.

[0269] In some embodiments, the two arms (or chains) of the constant region of the immunoglobulin heavy chain (e.g., an Fc polypeptide) can be heterodimerized by generating a "mortar and pestle" (KiH) mutation in the CH3 domain. This structural feature in the polypeptide arms allows the assembly of two half-antibodies (e.g., an Fc heterodimer; and a VH-CH domain and a VL-CL domain). For example, a heteromultimer (including a heterodimer) may comprise a first polypeptide and a second polypeptide, each containing a CH3 domain, wherein the two polypeptides interlock at an engineered interface within the CH3 domain, and the first polypeptide contains an engineered protrusion ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume larger than the original residue; and the second polypeptide contains an engineered cavity ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume smaller than the original residue. In some embodiments, the engineered interface of the heteromultimer comprises at least two pairs of protrusion-cavity mutants. The volume and accessible surface area of ​​each amino acid are described in AA Zamyatnin, Prog. Biophys., Mol. Biol. 24:107-123, 1972; and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, the lead-in residues used to form the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the lead-in residues used to form the cavity may be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residues used to form the cavity have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "groove" / protrusion chain and the T366S / L368A / Y407V mutation in the CH3 domain of the "mortar" / cavity chain. Additionally, the KiH conformation can be coupled with further mutations to allow SS disulfide bond linkage between the two chains. In this conformation, the protein / peptide is a heterodimer of the KiH conformation, with IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) linked to one and only one of two (or more) immunoglobulin heavy chain constant regions / chains (i.e., groove or mortar).

[0270] In some embodiments, two or more arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) may contain designs such as another symmetric-asymmetric stereocomplementary design (e.g., HA-TF, ZW1), charge-exchange interaction (DD-KK), charge-stereocomplementary exchange plus additional long-range electrostatic interaction (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic redirection, A107, Duobody, thereby forming a heterodimer / heteromultimer. Further descriptions of these conformations and exemplary mutations / residues can be found in FrontImmunol. 2016; 7: 394.

[0271] In another embodiment, the suitable protein or fragment thereof capable of translocating into the ER in the fusion protein is a globular protein, an immunoglobulin, or a fragment thereof. In various embodiments, the suitable protein or fragment thereof capable of translocating into the ER in the fusion protein is engineered with a signal peptide to translocate into a short polypeptide or protein in the ER. In some embodiments, the suitable protein or fragment thereof capable of translocating into the ER in the fusion protein is a short polypeptide or protein of about 2 kDa or no more than 250 kDa. As an example, short peptides or proteins are about 2 kDa to 5 kDa, about 6 kDa to 10 kDa, about 11 kDa to 20 kDa, about 21 kDa to 30 kDa, about 31 kDa to 40 kDa, about 41 kDa to 50 kDa, about 51 kDa to 75 kDa, about 76 kDa to 100 kDa, about 101 kDa to 125 kDa, about 126 kDa to 150 kDa, about 151 kDa to 175 kDa, about 176 kDa to 200 kDa, about 201 kDa to 225 kDa, or about 256 kDa to 250 kDa. As a further example, short peptides are flexible linkers or amino acid chains of 2, 3, or 4 amino acids, or 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, or 26 to 30 amino acids. As a further example, the short polypeptide is a flexible linker or amino acid chain of 1, 2, 3, or 4 amino acids, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acids. Other examples of these short polypeptides include, but are not limited to, dimers of two amino acids, trimers of three amino acids, tetramers of four amino acids, pentamers of five amino acids, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 235), and (GGGGX). λ (SEQ ID NO:236))n, where X λIt is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, such that the length of the amino acid linker is 25 amino acids or less. Other examples include (X λ GGGG (SEQ ID NO:317) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ GGG (SEQ ID NO:318) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ GG (SEQ ID NO:319) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGGX) λ G(SEQ ID NO:320)) n , where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5. Other examples include (X... λ GGG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ GG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGGX) λ )n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5. Other examples include (X... λ GG)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GGX) λ )n, where Xλ It is Q, A, E, or S, and n = 1 to 5, or in some implementations. Other examples include (X... λ G)n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5; (GX λ )n, where X λ It is Q, A, E, or S, and n = 1 to 5, or in some implementations, n is an integer greater than 5.

[0272] In another embodiment, suitable proteins capable of translocating in the ER may be globular proteins, human serum albumin (HSA), β2-microglobulin, transferrin, fragment antigen-binding regions (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, and fragments thereof. Other suitable proteins capable of translocating in the ER may include type I transmembrane proteins or fragments thereof, or type II transmembrane proteins or fragments thereof.

[0273] In various embodiments, the fusion protein comprising a short polypeptide or protein and IL-18 or a variant of IL-18 or a fragment thereof further comprises a second protein or a fragment thereof capable of translocating into the ER. The second protein or a fragment thereof capable of translocating into the ER may be an Fc domain or HSA, β2 microglobulin, transferrin, a fragment antigen-binding region (Fab region), a VHH antibody, a single-chain variable fragment (scFv), anticalin, a designed ankylosing repeat protein (DARPin), their binding domains and fragments thereof, or a type I transmembrane protein or a fragment thereof as described herein, or a type II transmembrane protein or a fragment thereof. Figure 50 A and Figure 53 A (the first three from left to right) are non-limiting examples of this type of fusion protein.

[0274] In other embodiments, the fusion protein also comprises a protein that cannot naturally translocate into the ER, such as a nuclear or cytoplasmic protein fused to the N-terminus of IL-18. For said protein, a signal peptide (which may be referred to as a leader sequence)—such as the Ig-κ leader sequence in FUSE-499 (e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO:247)), or one or more other signal peptides (including, but not limited to, those derived from human albumin and human azuril, see Kober et al., Biotechnol Bioeng. 2013 Apr; 110(4):1164-73)—is fused to the N-terminus of a non-ER translocation protein. For example, the signal peptide may be at the N-terminus of a propeptide or IL-18 (or a fragment, variant, or fragment variant thereof). Another example of a protein capable of translocating in / translocating to / across the ER may be a protein engineered with a signal peptide (e.g., at the N-terminus). As an example, Hsp70 is a nucleoprotein, but it can be engineered into an ER translocation protein when its N-terminus is fused or linked to a signal peptide. In various embodiments, the Fc, globular protein, or HSS originally present in the fusion proteins disclosed herein is replaced by the addition of an N-terminal signal peptide (such as an Ig-κ leader sequence).

[0275] In some implementations, the fusion protein (e.g., masked IL-18) also includes a tumor-targeting fragment, such as a fragment targeting cell surface proteins (including, but not limited to, tumor-associated antigens (TAAs)). Figure 51G The FUSE-517 shown is a masked IL-18 fusion protein that also contains an anti-EGFR antibody fragment, such as the Fab fragment of cetuximab. One or more antigen-targeting (preferably tumor antigen-targeting) fragments of known antibodies are considered compatible with the fusion protein system disclosed herein.

[0276] In some embodiments, the fusion protein (e.g., masked IL-18) comprises an activation receptor-targeting fragment, such as a fragment targeting activation receptors on the cell surface (including, but not limited to, CD16 on the surface of natural killer cells). The activation receptor-targeting fragment may be a fragment as described herein or a fragment known in the art as of the date of this filing. Activation receptors include immune receptor tyrosine-based activation motif (ITAM)-associated receptors, such as CD16 and NKp46. Activation receptors also include those involved in spontaneous NK cell activation, such as NKp46 (CD335), NKp30 (CD337), NKp44 (CD336), NKG2D (CD314), DNAM-1 (CD226), 2B4 (CD244), LFA-1 (CD11a-CD18), and CD2. In some embodiments, the fusion protein (e.g., 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, the CH2 domain of IgG1 and the CH2 domain of IgG4. In some embodiments, the fusion protein (e.g., masked IL-18) comprises a polypeptide fragment that targets immune checkpoints, such as fragments that target immune checkpoints expressed on T cells. For example, as Figure 23 The FUSE-694 shown is a masked IL-18 fusion protein that also contains an anti-PD1 fragment. Exemplary immune checkpoints include, but are not limited to, PD-1, PD-L1, CTLA-4, and LAG-3. One or more immune checkpoint targeting fragments of known antibodies are considered compatible with the fusion protein systems disclosed herein. Examples of anti-PD1 fragments include fragments (e.g., Fab, Fv) from pembrolizumab, nivolumab, pildolizumab, AMP-514, spartazumab, cimiprizumab, AK105, BCD-100, BI 754091, JS001, LZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tislelizumab, or PF-06801591. Other examples of anti-PD1 fragments include fragments (e.g., Fab, Fv) from vopalimumab, camrelizumab, sintilimab, AMP-224, AMP-514, and acrixolimab. Thus, for example, fusion proteins comprise, a timeline, an anti-PD1 fragment (e.g., Fab, Fv) from an anti-PD1 antibody, an IL-18 variant, and a protein capable of translocating into the ER (e.g., an Fc domain), and optionally, a linker.

[0277] In some embodiments, the fusion protein (e.g., masked IL-18) comprises a targeting peptide that targets a protein on the same surface as IL-18 RC. Examples of such proteins include, but are not limited to, CD16, γ9TCR, δ2 TCR or δ1 TCR, NKp46, CD137, CD40, or NKG2D. The peptide targeting CD16, γ9 TCR, δ2 TCR or δ1 TCR, NKp46, CD137, CD40, or NKG2D can be a peptide as described herein, or a peptide known in the art as of the date of this filing. In some embodiments, the fusion protein (e.g., masked IL-18) comprises a targeting peptide that targets a protein on a cell that does not contain IL-18RC. In these embodiments, the IL-18 fusion protein will need to be delivered close to the IL-18R complex to produce a cis or density effect, thereby facilitating the interaction between the IL-18 fusion protein and the IL-18R complex. For example, TAA-targeted IL-18 fusion proteins can interact with the IL-18R complex on T cells via: (a) the fusion protein bridging T cells to TAA+ cells; (b) the fusion protein combining with another protein bridging T cells to TAA+ cells; or (c) the fusion protein binding to TAA+ cells that natively interact with T cells via a second mechanism (e.g., TCR / MHC interaction). In other examples, the fusion protein can be delivered to fibroblasts or other helper cells in the tumor microenvironment and released by proteases, allowing it to act on distant IL-18R+ T cells or NK cells.

[0278] Exemplary targeting peptides include those peptides or fragments thereof shown in Table 18. Among those listed as antigen-binding antibodies, their VHH, Fab region, or single-chain variable fragment (scFv) can be used as antigen-binding sites for the multispecific antibodies disclosed herein.

[0279] 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 cleave the protein after the C-terminus of a lysine residue at its cleavage site Asp-Asp-Asp-Asp-Lys (SEQ ID NO:87). Therefore, the fragment produced by this cleavage reaction does not inherit any residues from the DDDDK (SEQ ID NO:87) recognition sequence. Additionally, DDDDK (SEQ ID NO:87) is part of an octapeptide FLAG tag (DYKDDDDK (SEQ ID NO:248)) and can be used as a fusion tag for antibody recognition, detection of the fusion protein by Western blot analysis, and purification of the fusion protein by anti-FLAG affinity chromatography.

[0280] Preferably, the cleavage site can be based on a peptide substrate sensitive to other enzymes, particularly proteases highly expressed in the tumor microenvironment, such as granzyme B, granzyme A, granzyme M, granzyme K, matrix metalloproteinases (MMPs) 1 / 2 / 9 / 14, or other MMPs. Notably, granzymes are typically upregulated only in inflamed tumors. For example, the substrate sequence of granzyme B could be Ile-Glu-Xaa-Asp↓Xaa-Gly (SEQ ID NO:249), wherein the cleavage occurs at the Asp↓Xaa peptide bond. Alternatively, the substrate sequence of granzyme B could also be Ile-Glu-Xaa-Asp↓, wherein the cleavage occurs at the C-terminus of Asp, and Xaa could be Gln (SEQ ID NO:88) or another amino acid.

[0281] Several immune cells, such as T cells, NK cells, neutrophils, and mast cells, can release granzymes. In several embodiments, fusion proteins comprising: (a) polypeptide fragments targeting immune checkpoints expressed on immune cells and / or polypeptide fragments targeting activating receptors on NK cells; and (b) tumor-targeting fragments that may induce the release of granzymes that release IL-18. For example, an IL-18 fusion protein comprising polypeptide fragments targeting immune checkpoint proteins can reverse the depletion of NK and / or T cells, thereby enabling these cells to release more granzymes.

[0282] In another embodiment, the fusion protein includes a cleavage site recognized by a serine protease, cysteine ​​protease, aspartic protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, or asparagine peptide lyase. In some embodiments, the protease cleavage site is recognized by the following proteases: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein (hK1, hK10, hK15), plasmin, collagenase, type IV collagenase, lysozyme, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase (caspase-3, Mir...). 1-CP), papain, HIV-1 protease, HSV protease, CMV protease, chymotrypsin, renin, pepsin, membrane protease, podocyte protein, Plasmodium protease, pitcher plant protease, exonuclease, endonuclease, matrix metalloproteinases (MMPs) (MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14), ADAM10, ADAM17, ADAM12, urokinase plasminogen activator The following are included: antiphospholipids (uPA), enterokinase, prostate-specific targets (PSA, hK3), interleukin-1β convertase, thrombin, FAP (FAP-α), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, protease 3, neutrophil serine protease 4, mast cell chymotrypsin, mast cell trypsin-like enzyme, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26). Non-limiting examples of cleavage sites are included in Table 6B. IEQD may be used as a specific example.

[0283] Variant, fragment, or fragment of a variant of IL-18 is considered suitable for the composition of the fusion protein and is preferred in some embodiments. For example, variants of mature IL-18 may have one, two, three, four, five, or more amino acid substitutions compared to wild-type mature IL-18. For example, one or more cysteine ​​residues in IL-18 or its propeptide may be replaced by natural or non-natural amino acids, such as from Cys to Ser, Ala, or Val, to reduce molecular aggregation in the fusion protein. Other 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.

[0284] Variants of IL-18 may have 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65%, or at least 60% sequence identity with wild-type IL-18. In some embodiments, variants of IL-18 may have at least 60% and at most 83% sequence identity with wild-type IL-18. In some embodiments, variants of IL-18 may have 60% to 64%, 65% to 69%, 70% to 74%, 75% to 89%, or 80% to 83% sequence identity with wild-type IL-18. In some embodiments, when cleaved at the cleavage site of the fusion protein, the IL-18 variant in the fusion protein is released as a functional fragment of approximately 15 kDa (e.g., on a tested electrophoresis gel). (It is speculated that the released protein may be mature IL-18, which normally has a molecular weight of 18 kDa, but may appear to be about 15 kDa due to the gradient used or a specific percentage of polyacrylamide in the gel.) Fragments of IL-18 may have 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65%, or at least 60% sequence identity (and / or length) with wild-type IL-18. In some embodiments, the IL-18 fragment generated from the fusion protein disclosed herein (especially after protease cleavage of the fusion protein) is less than 85% (e.g., about 83%, about 83% to 80%, about 80% to 75%, about 75% to 70%, or about 70% to 65%) the size of native / wild-type mature IL-18; for example, an IL-18 fragment of about 15 kDa (preferably having a binding affinity for IL-18Ra / b comparable to that of wild-type mature IL-18) is fused with a propeptide (or PP variant) and an ER translocation protein (with or without a mutation), and the fusion protein also contains a protease cleavage site such that, upon protease cleavage, a small IL-18 fragment (e.g., about 15 kDa) is released. Preferably, this small IL-18 fragment retains the native binding affinity for IL-18Ra / b and, relative to IL-18Ra / b, retains a binding affinity equal to or lower than that for IL-18BP. Preferably, the variant, fragment, or fragment of a variant of IL-18 is capable of binding to IL-18R and forming a complex, thereby activating the pro-inflammatory program and / or the NF-κB pathway. In some embodiments, the variant, fragment, or fragment of a variant of IL-18 is capable of having increased binding affinity (e.g., 150%, 140%, 130%, 120%, 110%, or at least 100% relative to wild-type IL-18) and / or inducing at least 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of biological activity relative to wild-type IL-18.In some embodiments, variants, fragments, or fragments of variants of IL-18 are capable of having a binding affinity increased by 120%, 110%, or at least 100% relative to wild-type IL-18, and / or inducing 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of biological activity relative to wild-type IL-18. In other embodiments, variants, fragments, or fragments of variants of IL-18 have reduced binding to IL-18-binding protein (IL-18BP) relative to wild-type IL-18.

[0285] In some embodiments, the biological activity of IL-18 cleaved from the fusion protein, or a fragment or variant thereof, is increased by at least 1,000-fold, 2,000-fold, 3,000-fold, 5,000-fold, 10,000-fold, 30,000-fold, 50,000-fold, 70,000-fold, 80,000-fold, 90,000-fold, or 100,000-fold compared to the uncleaved form in the fusion protein (especially one containing a propeptide). In further embodiments, the IL-18 cleaved from the fusion protein, or a fragment or variant thereof, has comparable biological activity, or a difference of about 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold in biological activity, compared to the mature recombinant human IL-18.

[0286] In some embodiments, IL-18 cleaved from the fusion protein, or a fragment or variant thereof, has a binding affinity to its IL-18R complex of about 18 nM (e.g., 18 nM ± 0.3 nM, 18 nM ± 0.5 nM, 18 nM ± 1.0 nM). In some embodiments, IL-18 cleaved from the fusion protein, or a fragment or variant thereof, has approximately the same or at least 100%, 95%, or 90% binding affinity to its IL-18R complex compared to wild-type IL-18. In some embodiments, the binding affinity of IL-18 cleaved from the fusion protein, or a fragment or variant thereof, to its IL-18R complex is greater than that of wild-type IL-18, for example, a binding affinity of at least 105%, 110%, or a KD value at least 10% or 20% lower than that of wild-type IL-18. Preferably, IL-18 cleaved from the fusion protein, or a fragment or variant thereof, has a reduced binding affinity for IL-18BP compared to wild-type IL-18. For example, in some cases, the KD of IL-18 cleaved from the fusion protein, or a fragment or variant thereof, with IL-18BP is 18 nM or higher, resulting in a lower binding affinity for IL-18BP than for IL-18R. In some cases, the KD of IL-18 cleaved from the fusion protein, or a fragment or variant thereof, with IL-18BP is 18 nM or higher, while the KD of wild-type IL-18 with IL-18BP is about 0.4 nM. It is also envisioned that the KD can vary depending on instrument and protocol settings.

[0287] In various embodiments, the fusion protein comprises a first polypeptide or protein or a fragment thereof capable of translocating into the endoplasmic reticulum (ER); and interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment thereof, wherein IL-18, the fragment of IL-18, the IL-18 variant, or a fragment thereof is located at the C-terminus 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 a wild-type IL-18 propeptide. In various embodiments, the protein or a fragment thereof capable of translocating into the endoplasmic reticulum (ER) is not a wild-type IL-18 propeptide. In addition to these features, other characteristics of the fusion protein are discussed herein.

[0288] In some embodiments, the fusion protein comprises an IL-18 variant. In various embodiments, the IL-18 variant has a composition comprising or derived from MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 10 ... The amino acid sequence consisting of amino acid positions 37-193 of SEQ ID NO:250 has one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250). In various embodiments, the one to five amino acid substitutions are 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 implementations, IL-18 variants contain no more than five amino acid substitutions, except for substituted cysteine.In various implementations, IL-18 variants have a composition containing or derived from MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID) The amino acid sequence consisting of amino acid positions 37-193 of SEQ ID NO:251 has one to five amino acid substitutions at positions E42, M87, K89, M96 and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).

[0289] In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251), with one or more amino acid substitutions at positions C74, C104, C112, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149.

[0290] In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 are each independently substituted with valine, alanine, or serine. In various embodiments, the one to five amino acid substitutions are one or more of the following: 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.

[0291] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 6B. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 6B further comprises a propeptide having an amino acid sequence selected from the propeptide column in Table 6B, and optionally from the same row as the IL-18 variant. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 6B and a 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 the propeptide. As a specific example, IEQD (SEQ ID NO: 88) may be used.

[0292] In various embodiments, the IL-18 variants are those disclosed in U.S. Patent 7,524,488, U.S. Patent Publication 2019 / 0070262, U.S. Patent Publication 2021 / 0015891, or PCT Publication WO 2022 / 038417, and the IL-18 variants and sequences of each of these patents or publications are incorporated herein by reference as if listed in their entirety.

[0293] In various embodiments, the fusion protein further comprises a targeting peptide. In some embodiments, the targeting peptide targets a protein on the cell surface, wherein the cell surface also has IL-18 RC or the cell is capable of expressing IL-18 RC. In various embodiments, the fusion protein binds to cells having IL-18 RC or capable of expressing IL-18 RC upon cell activation and activates IL-18 RC signaling.

[0294] In other embodiments, the targeting peptide targets proteins on the cell surface that do not possess IL-18 RC or cells that cannot express IL-18 RC. Cells without IL-18 RC or unable to express IL-18 RC on their cell surface are in close proximity to cells that express or are capable of expressing IL-18 RC. In other cases, the fusion protein can bring cells without IL-18 RC or unable to express IL-18 RC on their cell surface into close proximity to cells that express or are capable of expressing IL-18 RC.

[0295] In various implementation schemes, the targeting peptide includes a tumor-associated antigen-binding domain.

[0296] In various embodiments, the fusion protein further includes a binding domain of a protein expressed on an immune cell. In various embodiments, the fusion protein further includes a binding domain of a protein expressed on an immune cell expressing an IL-18 receptor complex or on an activated immune cell expressing an IL-18 receptor complex.

[0297] In various implementations, the fusion protein also comprises an antibody or antibody fragment. This allows the fusion protein to bind to tumor cells, or immune cells or stromal cells in tumor tissue, or tumor draining lymph nodes, or other secondary lymphoid organs. Examples of antibody fragments include Fc fragments, Fab fragments, Fv fragments, and other fragments discussed herein.

[0298] In various embodiments, the fusion protein also includes a masking domain. In some embodiments of these embodiments, the masking domain provides protection for IL-18, and IL-18 cannot be released by the protease. In some embodiments of these embodiments, mature IL-18 or a variant of mature IL-18 can be released from the masking domain by the protease. In various embodiments, the protease is a granzyme that can be released from immune cells. Examples of immune cells include, but are not limited to, NK cells, T cells, neutrophils, or mast cells. In various embodiments, the protease is a metalloproteinase that can be expressed in the tumor microenvironment or tumor-draining lymph nodes. Other examples of protease and granzyme types are described herein. In various embodiments, mature IL-18 increases the activity of NK cells or T cells, and optionally, the activity is one or more of proliferation, survival, and cytotoxicity.

[0299] In various embodiments, the fusion protein further comprises a half-life-extending molecule. Non-limiting examples of half-life-extending molecules are half-life-extending peptides; for example, human serum albumin (HSA) or an HSA-binding fragment. In various embodiments, when the fusion protein does not bind to cells possessing IL-18 RC, its activity is reduced compared to wild-type IL-18. In various embodiments, the reduced activity compared to wild-type IL-18 is at least a 75% reduction in activity.

[0300] In some implementations, the mature IL-18 variant at the C-terminus of the mask is attenuated. For example, mature IL-18-mut13A is attenuated to about 1 / 100.

[0301] In various embodiments, the fusion protein comprises peptide 1 and peptide 2 selected from Table 6A. In various embodiments, the fusion protein also comprises peptide 3 selected from Table 6A. In various embodiments, peptide 1 and peptide 2, as well as optional peptide 3, are selected from the same row of Table 6A.

[0302] In various embodiments, the fusion protein comprises polypeptide 1 selected from Table 6A, wherein polypeptide 1 comprises HSA.

[0303] In various embodiments, the fusion protein does not contain any of the IL-18 variants disclosed in U.S. Patent 7,524,488, U.S. Patent Publication 2019 / 0070262, U.S. Patent Publication 2021 / 0015891, or PCT Publication WO 2022 / 038417, the IL-18 variants and sequences of each of these patents or publications being incorporated herein by reference as if fully listed.

[0304] Various embodiments of the present invention include propeptide variants. In various embodiments, the propeptide variants have the following amino acid sequence: AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, wherein X1 is any amino acid other than cysteine ​​(SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76).

[0305] Various embodiments include IL-18 variants for the multi-cytokine fusion protein 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 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250). In various embodiments, the one to five amino acid substitutions are 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 variants contain no more than five amino acid substitutions, except for substituted cysteine.

[0306] In various implementations, IL-18 variants have a composition containing or derived from MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID) The amino acid sequence consisting of amino acid positions 37-193 of SEQ ID NO:251 has one to five amino acid substitutions at positions E42, M87, K89, M96 and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).

[0307] In various implementations, IL-18 variants have a composition containing or derived from MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID) The amino acid sequence consisting of positions 37-193 of SEQ ID NO:251 has one or more amino acid substitutions at positions C74, C104, C112, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149. In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 are each independently substituted with valine, alanine, or serine.

[0308] In various embodiments, the one to five amino acid substitutions are one or more of the following: 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.

[0309] In various implementations, the IL-18 variants are selected from the “Mature IL18 Variants” column in Table 6B.

[0310] In various implementations, IL-18 variants also include IL-18 propeptide or IL-18 propeptide variants.

[0311] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant having the following amino acid sequence: AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, where X1 is any amino acid other than cysteine ​​(SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76). In various embodiments, the IL-18 variant selected from the "Mature IL18 Variants" column of Table 6B further comprises a propeptide having an amino acid sequence selected from the "Propeptide" column of Table 6B, and optionally from the same row as the IL-18 variant.

[0312] In various embodiments, the IL-18 variants also include an IL-18 propeptide variant and a cleavage peptide. In various embodiments, the cleavage peptide is selected from Table 6B. As a specific example, IEQD can be used.

[0313] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 6B, a propeptide selected from Table 6B, and a cleavage peptide selected from Table 6B, and optionally, the cleavage peptide comes from the same row as the IL-18 variant and the propeptide.

[0314] In various embodiments, the IL-18 variants are not those disclosed in U.S. Patent 7,524,488, U.S. Patent Publication 2019 / 0070262, U.S. Patent Publication 2021 / 0015891, or PCT Publication WO 2022 / 038417, the IL-18 variants and sequences of each of these patents or publications being incorporated herein by reference as if fully listed.

[0315] Table 6A. Amino acid sequences of each component in an exemplary fusion protein ("FUSE"). Some sequences from the "peptide 1" sequence. The first polypeptide arm sequence is located at FUSE-422, FUSE-423, FUSE-424, FUSE-441, FUSE-442, and 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, FUSE-547, FUSE5-556, FUSE-583-587, FUSE- 599-602, FUSE-645, FUSE-686, FUSE-756-758, FUSE-775, FUSE-874-876 and FUSE-878-892 They are the same .

[0316] For FUSE1179 and 1229: (1) Regular font = short peptide; (2) Italic bold font = IL-18 variant; (3) Italic font = Fc epsilon; (4) Italic and underlined font = Fc gamma; (5) Bold font = Fab polypeptide 3 sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:73) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE (SEQ ID NO:74) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO:75) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:280) DIVMTQGTLPNPPVSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:281) GGGGSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNEDGGGGSGGGGGSGGGSGGGSSDIVMTQGTLPNPVPSGESVS ITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. ID NO:282) Table 7. Nucleotide sequences encoding the corresponding polypeptides in Table 6A .

[0317] Table 20. Yield of exemplary fusion proteins .

[0318] ROR1 Details and Examples The contents of international application No. PCT / US2023 / 070587, filed on July 20, 2023, entitled “Receptor Tyrosine Kinase-Like OrphanReceptor I (ROR1)-Specific VHH Antibodies and Multispecific Antibodies Thereof as Immune Cell Engagers,” are incorporated herein by reference as if they were fully disclosed.

[0319] This document discloses immune-specific antibodies (e.g., isolated antibodies) or antigen-binding fragments thereof that can be used with the multi-cytokine fusion protein of the present invention to bind to ROR1. Multi-specific antibody constructs that can be used in methods involving the administration of a separate fusion protein / construct are also described.

[0320] The inventors have identified human ROR1-specific VHHs generated in camels in response to human ROR1 stimulation (therefore referred to as Bactrian camel-derived VHHs). These human ROR1-stimulated camel-derived VHHs include: 2A11 (a polypeptide comprising the amino acid sequence QVQLQESGGGSVPAGGSLRLSCAASGSTYSANCMGWFRQAPGKEREEVASMSIRSGRTYYSDSVKGRFTISQDGSKNTLYLQLNSLKAEDTALYYCAAAYGGSRCVYNYRGQGTQVTVSS (SEQ ID NO:331)), and 5A1 (a polypeptide comprising the amino acid sequence QVQLQESGGGSVQAGGSLKLSCTASGYTNRLKCMGWFRQAPGKEREEIATISTGTGNTYYADSVKGRFTFSQDKVKNTVYLQMNTLKPDDTGMYYCAADVRPDGTTCHYNSGGQGTQVTVSS (SEQ ID NO:332)).

[0321] The isolated anti-ROR1 (or ROR1-specific) antibody or its ROR1-binding fragment may contain 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 may 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 complementarity-determining regions (CDRs) for binding human ROR1, and these CDRs are recognized / present in 2A11, wherein CDR1 of 2A11 consists of a polypeptide having the amino acid sequence GSTYSANC (SEQ ID NO:325), CDR2 of 2A11 consists of a polypeptide having the amino acid sequence MSIRSGRTY (SEQ ID NO:326), and CDR3 of 2A11 consists of a polypeptide having the amino acid sequence AYGGSRCVYNY (SEQ ID NO:327).

[0323] In some embodiments, a polypeptide is provided comprising one or more of the following: a variant of the polypeptide having the amino acid sequence of SEQ ID NO:325, a variant of the polypeptide having the amino acid sequence of SEQ ID NO:326, and a variant of the polypeptide having the amino acid sequence of SEQ ID NO:327, wherein each of these variants comprises one or more deletions, additions, or substitutions of amino acid residues compared to the corresponding “wild-type” polypeptide having the amino acid sequences of SEQ ID NO:325, SEQ ID NO:326, or SEQ ID NO:327, respectively. In various embodiments, the substitution is a conservative substitution. Preferably, the variant of the polypeptide having the amino acid sequence of SEQ ID NO:325, the variant of the polypeptide having the amino acid sequence of SEQ ID NO:326, and / or the variant of the polypeptide having the amino acid sequence of SEQ ID NO:327 is capable of binding human ROR1, more preferably, is also capable of immune-specific binding to human ROR1, or has a binding affinity for ROR1 of at least 90%, 80%, 70%, 60%, or 50% compared to the corresponding “wild-type” CDR polypeptide present in 2A11. Preferably, in the polypeptide that is a variant of 2A11, or in the polypeptide that is a variant of the polypeptide having the amino acid sequence of SEQ ID NO:325, SEQ ID NO:326, or SEQ ID NO:327, one or both cysteine ​​residues in the CDR of 2A11 (i.e., the cysteine ​​residues in SEQ ID NO:325 and SEQ ID NO:327) remain unchanged. Therefore, in some embodiments, polypeptides including one or more of the following: polypeptide variants having the amino acid sequence of SEQ ID NO:325, polypeptide variants having the amino acid sequence of SEQ ID NO:326, and polypeptide variants having the amino acid sequence of SEQ ID NO:327, also retain those cysteine ​​residues as in the polypeptide having the amino acid sequence of SEQ ID NO:325 and the polypeptide having the amino acid sequence of SEQ ID NO:327. Alternatively, the polypeptide can be a variant of 2A11 in which one or both cysteine ​​residues in the CDR of 2A11 are replaced by a native or non-native amino acid containing a cross-linking functional group, thereby promoting the formation of intermolecular / intramolecular bonds; examples of such amino acids include, but are not limited to, p-benzoyl-L-phenylalanine (Bpa), azides, and alkyl halides. The ability to maintain disulfide bonds and / or intramolecular bonds at those residue positions in the CDR of 2A11 where cysteine ​​is present is considered crucial because Figure 62G The data indicate that replacing cysteine ​​residues with valine significantly weakens the binding affinity of 2A11 to the antigen.

[0324] In some embodiments, amino acids in the non-CDR portion of 2A11 are modified to produce a humanized form of 2A11 (containing the CDRs of SEQ ID NO:325, SEQ ID NO:326, and SEQ ID NO:327, as well as the humanized frame / non-CDR portion), thereby making it more suitable for human subjects. Therefore, various embodiments provide a humanized anti-ROR1 antibody or a fragment thereof.

[0325] In another embodiment, an anti-ROR1 (or ROR1-specific) heavy chain antibody is provided, comprising (1) an antigen-binding moiety comprising a VHH of a polypeptide having the amino acid sequence of SEQ ID NO:331, or a VHH of a polypeptide having the amino acid sequences of SEQ ID NO:325, SEQ ID NO:326, and SEQ ID NO:327; and (2) an Fc domain of an Ig heavy chain. Preferably, the heavy chain antibody further comprises a hinge domain of an Ig heavy chain such that the VHH is linked to the hinge and Fc domain of the Ig heavy chain. The disclosed anti-ROR1 heavy chain antibody or its antigen-binding fragment includes all isotypes, namely IgA, IgD, IgE, IgG, and IgM, as well as synthetic multimers of a four-chain immunoglobulin (Ig) structure, and the IgY isotype commonly found in hen or turkey serum and hen or turkey egg yolks. In some embodiments, the anti-ROR1 heavy chain antibody comprises (1) an antigen-binding portion, which is a VHH comprising a polypeptide having the amino acid sequence of SEQ ID NO:331, or a VHH comprising a CDR having the amino acid sequences of SEQ ID NO:325, SEQ ID NO:326 and SEQ ID NO:327; and (2) a hinge domain and an Fc domain of an IgG heavy chain, wherein the IgG may be any one of the subclasses IgG1, IgG2, IgG3 and IgG4.

[0326] The isolated anti-ROR1 (or ROR1-specific) antibody or its ROR1-binding fragment may optionally contain 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 may comprise or consist of a polypeptide having the amino acid sequence of SEQ ID NO:331.

[0327] In some embodiments, a polypeptide is provided comprising one or more complementarity-determining regions (CDRs) of 5A1, wherein CDR1 of 5A1 consists of a polypeptide having the amino acid sequence GYTNRLKC (SEQ ID NO:328), CDR2 of 5A1 consists of a polypeptide having the amino acid sequence ISTGTGNTY (SEQ ID NO:329), and CDR3 of 5A1 consists of a polypeptide having the amino acid sequence DVRPDGTTCHYNS (SEQ ID NO:330).

[0328] In some embodiments, a polypeptide is provided comprising one or more of the following: 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 the amino acid sequence of SEQ ID NO:330, wherein each of these variants comprises one or more deletions, additions, or substitutions of amino acid residues compared to the corresponding “wild-type” polypeptide having the amino acid sequence of SEQ ID NO:328, SEQ ID NO:329, or SEQ ID NO:330. In various embodiments, the substitution is a conservative substitution. Preferably, the variant of the polypeptide having the amino acid sequence of SEQ ID NO:328, the variant of the polypeptide having the amino acid sequence of SEQ ID NO:329, and / or the variant of the polypeptide having the amino acid sequence of SEQ ID NO:330 is capable of binding human ROR1, more preferably, is also capable of immune-specific binding to human ROR1, or has a binding affinity for human ROR1 of at least 90%, 80%, 70%, 60%, or 50% compared to the corresponding “wild-type” CDR present in 5A1. Preferably, in the polypeptide that is a variant of 5A1, or in the polypeptide that is a variant of the polypeptide having the amino acid sequence of SEQ ID NO:328, SEQ ID NO:329, or SEQ ID NO:330, one or both cysteine ​​residues in the CDR of 5A1 (i.e., the cysteine ​​residues in SEQ ID NO:328 and SEQ ID NO:330) remain unchanged. Therefore, in some embodiments, polypeptides comprising one or more of the variants of SEQ ID NO:328, SEQ ID NO:329, and SEQ ID NO:330 also retain those cysteine ​​residues as in SEQ ID NO:328 and SEQ ID NO:330. Alternatively, the polypeptide can be a variant of 5A1 in which one or both cysteine ​​residues of the CDR of 5A1 are replaced by a native or non-native amino acid containing a cross-linking functional group, thereby promoting the formation of intermolecular / intramolecular bonds; examples of such amino acids include, but are not limited to, p-benzoyl-L-phenylalanine (Bpa), azides, and haloalkanes. The ability to maintain disulfide bonds and / or intramolecular bonds at these residue positions is considered crucial because Figure 62F The data indicate that replacing cysteine ​​residues with valine significantly weakens the binding affinity of 5A1 to the antigen.

[0329] In some implementations, amino acids in the non-CDR portion of 5A1 are modified to produce a humanized form of 5A1 (comprising a CDR with amino acid sequences SEQ ID NO:328, SEQ ID NO:329, and SEQ ID NO:330, and a humanized frame / non-CDR portion), thereby making it better suited for human subjects. An exemplary humanized VHH derived from 5A1 includes: 5A1-H1 (a polypeptide having the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGYTNRLKCMGWFRQAPGKERELASISTGTGNTYYADSVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCAADVRPDGTTCHYNSRGQGTLVTVSS (SEQ ID NO:335)). 5A1-H2 (a polypeptide having the amino acid sequence QVQLQESGGGLVQPGGSLRLSCTASGYTNRLKCMGWVRQAPGKEREEVATISTGTGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAADVRPDGTTCHYNSGGQGTQVTVSS (SEQ ID NO:336)). 5A1-H3 (a polypeptide having the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGYTNRLKCMGWFRQAPGKEREEVSTISTGTGNTYYADSVKGRFTISQDKSKNTLYLRMNSLRAEDTALYYCAADVRPDGTTCHYNSGGQGTQVTVSS (SEQ ID NO:337)). 5A1-H4 (a polypeptide having the amino acid sequence EVQLLESGGGLVPRGGSLRLSCTASGYTNRLKCMGWFRQAPGKEREEIATISTGTGNTYYADSVKGRFTISRDNSRNTLYLQMKTLRAEDTAVYYCAADVRPDGTTCHYNSWGQGTQVTVSS (SEQ ID NO:338)), and 5A1-H5 (a polypeptide having the amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGYTNRLKCMGWFRQAPGKEREEIATISTGTGNTYYADSVKGRFTFSRDNSKNTLYLQMNSLRAEDTAVYYCAADVRPDGTTCHYNSGGQGTQVTVSS (SEQ ID NO:339)).

[0330] Other exemplary humanized VHHs are shown in Table 14.

[0331] In another embodiment, an anti-ROR1 (or ROR1-specific) heavy chain antibody is provided, the heavy chain antibody comprising (1) an antigen-binding moiety, which is (i) a VHH comprising a polypeptide having the amino acid sequence of SEQ ID NO:332, or (ii) a VHH comprising CDRs having the amino acid sequences of SEQ ID NO:328, SEQ ID NO:329, and SEQ ID NO:330, or (iii) a VHH comprising a polypeptide having the amino acid sequences of any one of SEQ ID NO:335 to SEQ ID NO:339, or (iv) comprising one or more of (i), (ii), and (iii); and (2) an Fc domain of an Ig heavy chain. Preferably, the heavy chain antibody further comprises a hinge domain of an Ig heavy chain, such that the VHH is linked to the hinge and Fc domain of the Ig heavy chain. The disclosed anti-ROR1 heavy chain antibody or its antigen-binding fragment includes all isotypes, namely IgA, IgD, IgE, IgG, and IgM, as well as synthetic multimers with a four-chain immunoglobulin (Ig) structure, and the IgY isotype commonly found in hen or turkey serum and hen or turkey egg yolks. In some embodiments, the anti-ROR1 heavy chain antibody comprises (1) an antigen-binding moiety that is a VHH having the amino acid sequence of SEQ ID NO:332, or a VHH containing a CDR having the amino acid sequences of SEQ ID NO:328, SEQ ID NO:329, and SEQ ID NO:330, or a VHH having the amino acid sequences of any one of SEQ ID NO:335 to SEQ ID NO:339; and (2) a hinge domain and an Fc domain of an IgG heavy chain, wherein the IgG may be any one of subclasses IgG1, IgG2, IgG3, and IgG4.

[0332] A polypeptide is also provided, comprising (1) two or more repeating sequences of a polypeptide having the amino acid sequence of SEQ ID NO:331, (2) two or more repeating sequences of a polypeptide having the amino acid sequence of SEQ ID NO:332, (3) one or more repeating sequences of a polypeptide having the amino acid sequence of SEQ ID NO:331 and one or more repeating sequences of a polypeptide having the amino acid sequence of SEQ ID NO:332, (4) two or more repeating sequences of a polypeptide having the amino acid sequence of any one of SEQ ID NO:335 to SEQ ID NO:339, or (5) one or more repeating sequences of a first polypeptide having the amino acid sequence of one of SEQ ID NO:335 to SEQ ID NO:339 and one or more repeating sequences of a second polypeptide having the amino acid sequence of one of SEQ ID NO:335 to SEQ ID NO:339, wherein the first polypeptide and the second polypeptide have different amino acid sequences. In some embodiments, the polypeptide comprises tandem sequences of a first VHH and a second VHH, wherein the first VHH and the second VHH may be the same or different. The amino acid sequences of the first VHH and the second VHH can be selected from either SEQ ID NO:331 and SEQ ID NO:332, or from either SEQ ID NO:335 to SEQ ID NO:339. In one aspect, the first VHH and the second VHH are identical and both have the amino acid sequence of SEQ ID NO:331; in another aspect, the first VHH and the second VHH are identical 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 a polypeptide having the 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 a 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 a 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 a polypeptide having the amino acid sequence of SEQ ID NO:332. In a further embodiment, the polypeptide comprising the tandem sequence further comprises a linker, wherein the first VHH and the second VHH are connected to the linker. The connector can be located on the N end of VHH, which is closer to the N end of Fc, or alternatively, on the C end.

[0333] Various embodiments provide a polypeptide comprising: a polypeptide having a complementarity-determining region (CDR) 1 selected from Table 15A, a polypeptide having a CDR2, and a polypeptide having a CDR3, wherein CDR1, CDR2, and CDR3 are selected from the same row in Table 15A. Various embodiments provide a polypeptide comprising: a polypeptide having a complementarity-determining region (CDR) 1 selected from Table 15B, a polypeptide having a CDR2 selected from Table 15B, 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 a polypeptide comprising variants of a polypeptide having: a polypeptide having CDR1 selected from Table 15A, a polypeptide having CDR2 selected from Table 15A, and a polypeptide having CDR3 selected from Table 15A, wherein the variant of the polypeptide having CDR1 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR1, wherein the variant of the polypeptide having CDR2 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR2, and wherein the variant of the polypeptide having CDR3 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR3, wherein CDR1, CDR2, and CDR3 are selected from the same row in Table 15A. Various embodiments provide a polypeptide comprising variants of a polypeptide having: a polypeptide having CDR1 selected from Table 15B, a polypeptide having CDR2 selected from Table 15B, and a polypeptide having CDR3 selected from Table 15B, wherein the variant of the polypeptide having CDR1 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR1, wherein the variant of the polypeptide having CDR2 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR2, and wherein the variant of the polypeptide having CDR3 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having CDR3, wherein CDR1, CDR2, and CDR3 are selected from the same row in Table 15B. Various embodiments also provide a polypeptide comprising: a polypeptide having CDR1 having SEQ ID NO:328, a polypeptide having CDR2 having SEQ ID NO:401, and a polypeptide having CDR3 having SEQ ID NO:330. Various embodiments provide a polypeptide comprising: a polypeptide having CDR1 of SEQ ID NO:397, a polypeptide having CDR2 of SEQ ID NO:401, and a polypeptide having CDR3 of SEQ ID NO:408. Various embodiments provide a polypeptide comprising: a polypeptide having CDR1 of SEQ ID NO:397, a polypeptide having CDR2 of SEQ ID NO:401, and a polypeptide having CDR3 of SEQ ID NO:409.Various embodiments provide a polypeptide comprising: a polypeptide having CDR1 of SEQ ID NO:397, a polypeptide having CDR2 of SEQ ID NO:329, and a polypeptide having CDR3 of SEQ ID NO:408. Various embodiments provide a polypeptide comprising: a polypeptide having CDR1 of SEQ ID NO:397, a polypeptide having CDR2 of SEQ ID NO:329, and a polypeptide having CDR3 of SEQ ID NO:409. In various embodiments, the substitution is a conservative substitution. Preferably, variants of CDR1, CDR2, or CDR3 are capable of binding human ROR1; more preferably, they are also capable of immune-specific binding to human ROR1, or have a binding affinity for ROR1 of at least 90%, 80%, 70%, 60%, or 50% compared to the corresponding CDR polypeptide. In various embodiments, the polypeptide further comprises frame regions (FWRs) 1, 2, 3, and 4 selected from Table 15B, wherein FWR1, FWR2, FWR3, and FWR4 are selected from the same row in Table 15B. In various embodiments, polypeptides having CDR1, CDR2, and CDR3 further comprise FWR1, FWR2, FWR3, and FWR4 derived from human IgG. In various embodiments, polypeptides having variants of CDR1, CDR2, and CDR3 further comprise FWR1, FWR2, FWR3, and FWR4 derived from human IgG. In various embodiments, the polypeptide is a VHH 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 substitutions of amino acid residues of the polypeptide. In various embodiments, the variant comprises up to five deletions, additions, or substitutions of amino acid residues of the polypeptide. In various embodiments, the variant comprises up to one, two, three, or four deletions, additions, or substitutions of amino acid residues of the polypeptide. In various embodiments, the variant is at least 95% identical to the polypeptides selected from Table 14. In various embodiments, the variant is at least 96%, 97%, 98%, or 99% identical to the polypeptides 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 the corresponding polypeptides selected from Table 14. In various embodiments, the polypeptide comprises a VHH polypeptide having the 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 polypeptide is a variant of a VHH polypeptide having the 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 substitutions of amino acid residues of the polypeptide.In various embodiments, the variant comprises up to five deletions, additions, or substitutions of amino acid residues of the polypeptide. In various embodiments, the variant comprises up to one, two, three, or four deletions, additions, or substitutions of amino acid residues of the polypeptide. In various embodiments, the variant is at least 95% identical to a VHH polypeptide having the 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 96%, 97%, 98%, or 99% identical to a VHH polypeptide having the 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 a VHH polypeptide having the amino acid sequence of SEQ ID NO:340, SEQ ID NO:384, SEQ ID NO:363, or SEQ ID NO:364.

[0334] The isolated antibody or its antigen-binding fragment can preferably bind to the region of the ROR1 extracellular domain, and thus bind to cells with ROR1 surface expression.

[0335] As disclosed above, the anti-ROR1 VHH (nanobody), heavy chain antibody, and / or its antigen-binding fragment, or peptide containing anti-ROR1 VHH or its CDR, can be derived from any species through recombinant means. For example, the VHH, heavy chain antibody, or antigen-binding fragment can be mouse, rat, goat, horse, pig, cattle, chicken, rabbit, camel, donkey, human, or a chimeric form thereof. In particular, the VHH, heavy chain antibody, or antigen-binding fragment can be camel or its humanized form. To suit human administration, non-human antibodies or antigen-binding fragments may undergo genetic or structural alterations to reduce their antigenicity when administered to human patients.

[0336] Also provided are polynucleotide sequences encoding the disclosed anti-ROR1 VHH, the disclosed anti-ROR1 heavy chain antibody, and / or the disclosed polypeptide containing anti-ROR1 VHH or its CDR. For example, vectors encoding sequences encoding 2A11, 5A1, or humanized forms of 2A11 or 5A1 can be expressed in a variety of transfection-compatible cell types, not limited to camel cells or human cells. Furthermore, vectors encoding any of the sequences shown in Table 14 can be expressed in a variety of transfection-compatible cell types, not limited to camel cells or human cells. Vectors containing polynucleotides are also provided. These vectors can be expression vectors, such as recombinant expression vectors. Expression vectors may contain one or more additional sequences, such as, but not limited to, regulatory sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a variety of host cells are well known and include, but are not limited to, plasmids, phage particles, phages, baculoviruses, rod particles, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors. The invention also provides cells capable of expressing the disclosed vectors. These cells can be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as E. coli). The disclosed antibodies can also be produced by hybridoma cells.

[0337] Multispecific antibodies and multispecific antigen-binding fragments This document also discloses multispecific (including bispecific or trispecific) antibodies or multispecific antigen-binding fragments thereof that bind to at least ROR1 and CD3 (anti-ROR1 / anti-CD3 antibody), or bind to at least ROR1 and an activating receptor expressed on immune cells (including, but not limited to, CD3, CD16, γ9 TCR, δ2 TCR or δ1 TCR, NKp46, and NKG2D). In various embodiments, isolated bispecific antibodies or bispecific antigen-binding fragments thereof that bind to ROR1 and CD3 are provided (anti-ROR1 / anti-CD3 bispecific antibody).

[0338] In various embodiments, the anti-ROR1 / anti-CD3 antibody has at least a first antigen-binding site (ROR1 arm) that specifically binds to ROR1 and a second antigen-binding site (CD3 arm) that specifically binds to CD3. Exemplary anti-ROR1 / anti-CD3 antibodies of these embodiments are shown in [illustration details missing]. Figure 58A and Figure 58E middle.

[0339] In some embodiments, the anti-ROR1 / anti-CD3 antibody has at least a ROR1-specific first arm and a bispecific second arm, wherein the ROR1-specific first arm contains a first antigen-binding site that specifically binds to ROR1, and the bispecific second arm contains an antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to ROR1. Exemplary anti-ROR1 / anti-CD3 antibodies of these embodiments are shown in [illustration details missing]. Figure 58B and Figure 58C middle.

[0340] In various implementations, the anti-ROR1 / anti-NKG2D antibody has at least a first antigen-binding site (ROR1 arm) that specifically binds to ROR1 and a second antigen-binding site (NKG2D arm) that specifically binds to NKG2D.

[0341] In some embodiments, the anti-ROR1 / anti-NKG2D antibody has at least a ROR1-specific first arm and a bispecific second arm, wherein the ROR1-specific first arm contains an immune-specific first antigen-binding site for binding ROR1, and the bispecific second arm contains an immune-specific antigen-binding site for binding NKG2D and an immune-specific second antigen-binding site for binding ROR1.

[0342] In various embodiments, the anti-ROR1 / anti-CD16 antibody has at least a first antigen-binding site (ROR1 arm) that specifically binds to ROR1 and a second antigen-binding site (CD16 arm) that specifically binds to CD16.

[0343] In some embodiments, the anti-ROR1 / anti-CD16 antibody has at least a ROR1-specific first arm and a bispecific second arm, wherein the ROR1-specific first arm contains a first antigen-binding site that specifically binds to ROR1, and the bispecific second arm contains an antigen-binding site that specifically binds to CD16 and a second antigen-binding site that specifically binds to ROR1.

[0344] In various implementations, the anti-ROR1 / anti-NKp46 antibody has at least a first antigen-binding site (ROR1 arm) that specifically binds to ROR1 and a second antigen-binding site (NKp46 arm) that specifically binds to NKp46.

[0345] In some embodiments, the anti-ROR1 / anti-NKp46 antibody has at least a ROR1-specific first arm and a bispecific second arm, wherein the ROR1-specific first arm contains an immune-specific first antigen-binding site for binding ROR1, and the bispecific second arm contains an immune-specific antigen-binding site for binding NKp46 and an immune-specific second antigen-binding site for binding ROR1.

[0346] In a further embodiment, an isolated multispecific antibody or a multispecific antigen-binding fragment thereof is provided, comprising at least one ROR1 binding site, at least one CD3 binding site, and an Fc domain of an Ig heavy chain, and optionally a hinge domain of an Ig heavy chain. The Ig heavy chain can be any of the following: isotypes IgA, IgD, IgE, IgG, and IgM, as well as synthetic polymers of a four-stranded immunoglobulin (Ig) structure, and the IgY isotype commonly found in hen or turkey serum and hen or turkey egg yolks. Preferably, the Ig heavy chain is an IgG heavy chain, which can be any of the subclasses IgG1, IgG2, IgG3, and IgG4.

[0347] Therefore, in some embodiments, the anti-ROR1 / anti-CD3 antibody has at least (1) a first antigen-binding site specific to ROR1, (2) a second antigen-binding site specific to CD3, or a bispecific antigen-binding site specific to both ROR1 and CD3, and (3) hinge domains and Fc domains of two or more chains of Ig, which may be dimer or polymeric, wherein at least one heavy chain of Ig may also contain an antigen-binding site specific to ROR1, preferably located at the C-terminus of the Ig heavy chain.

[0348] Therefore, in other embodiments, the anti-ROR1 / anti-NKG2D antibody has at least (1) a first antigen-binding site specific to ROR1, (2) a second antigen-binding site specific to NKG2D, or a bispecific antigen-binding site specific to both ROR1 and NKG2D, and (3) hinge domains and Fc domains of two or more chains of Ig, which may be dimer or polymeric, wherein at least one heavy chain of Ig may also contain an antigen-binding site specific to ROR1, preferably located at the C-terminus of the Ig heavy chain.

[0349] Therefore, in some other embodiments, the anti-ROR1 / anti-CD16 antibody has at least (1) a first antigen-binding site specific to ROR1, (2) a second antigen-binding site specific to CD16, or a bispecific antigen-binding site specific to both ROR1 and CD16, and (3) hinge domains and Fc domains of two or more chains of Ig, which may be dimer or polymeric, wherein at least one heavy chain of Ig may also contain an antigen-binding site specific to ROR1, preferably located at the C-terminus of the Ig heavy chain.

[0350] Therefore, in other embodiments, the anti-ROR1 / anti-NKp46 antibody has at least (1) a first antigen-binding site specific to ROR1, (2) a second antigen-binding site specific to NKp46, or a bispecific antigen-binding site specific to both ROR1 and NKp46, and (3) hinge domains and Fc domains of two or more chains of Ig, which may be dimer or polymeric, wherein at least one heavy chain of Ig may also contain an antigen-binding site specific to ROR1, preferably located at the C-terminus of the Ig heavy chain.

[0351] Alternatively, an isolated multispecific antibody or a multispecific antigen-binding fragment thereof is provided, comprising at least one ROR1 binding site, at least one CD3 binding site, and a scaffold protein, such as human serum albumin. Alternatively, an isolated multispecific antibody or a multispecific antigen-binding fragment thereof is provided, comprising at least one ROR1 binding site, at least one NKG2D binding site, and a scaffold protein, such as human serum albumin. Alternatively, an isolated multispecific antibody or a multispecific antigen-binding fragment thereof is provided, comprising at least one ROR1 binding site, at least one NKp46 binding site, and a scaffold protein, such as human serum albumin. Alternatively, an isolated multispecific antibody or a multispecific antigen-binding fragment thereof is provided, comprising at least one ROR1 binding site, at least one NKp46 binding site, and a scaffold protein, such as human serum albumin.

[0352] Isolated multispecific antibodies or their multispecific antigen-binding fragments may contain: a) An immune-specific binding site for ROR1, wherein the first antigen-binding site comprises one or more of the following: 2A11 (This peptide has the amino acid sequence of SEQ ID NO:331). The variants of 2A11 disclosed above, 5A1 (this peptide has the amino acid sequence of SEQ ID NO:332). Variants of the 5A1 disclosed above (such as polypeptides having the amino acid sequence of any one of SEQ ID NO:335 to SEQ ID NO:339). Three, two, or at least one of the following amino acids from 2A11: CDR1 (having the amino acid sequence of SEQ ID NO:325), CDR2 (having the amino acid sequence of SEQ ID NO:326), and CDR3 (having the amino acid sequence of SEQ ID NO:327). One or more variants of three, two, or at least one of the following: CDR1 (having the amino acid sequence of SEQ ID NO:325), CDR2 (having the amino acid sequence of SEQ ID NO:326), and CDR3 (having the amino acid sequence of SEQ ID NO:327) of 2A11. Three, two, or at least one of the following amino acids from 5A1: CDR1 (having the amino acid sequence of SEQ ID NO:328), CDR2 (having the amino acid sequence of SEQ ID NO:329), and CDR3 (having the amino acid sequence of SEQ ID NO:330). One or more variants of three, two, or at least one of the following: CDR1 (having the amino acid sequence of SEQ ID NO:328), CDR2 (having the amino acid sequence of SEQ ID NO:329), and CDR3 (having the amino acid sequence of SEQ ID NO:330); and b) The second antigen binding site that specifically binds to CD3, CD16, NKp46 or NKG2D.

[0353] In various embodiments, the second antigen-binding site immune-specifically binds to CD3. In various embodiments, the antigen-binding site immune-specifically binds to NKG2D.

[0354] In other embodiments, the isolated multispecific antibody or its multispecific antigen-binding fragment may comprise: a1) Contains a first antigen-binding site of a polypeptide selected from Table 14, or a2) Contains the first antigen binding site selected from CDR1, CDR2, and CDR3 in Table 15A, or a3) Contains the first antigen binding sites selected from CDR1, CDR2, and CDR3 in Table 15B; and b) The second antigen binding site that specifically binds to CD3, CD16, NKp46 or NKG2D.

[0355] In various embodiments, the second antigen-binding site immune-specifically binds to CD3. In various embodiments, the antigen-binding site immune-specifically binds to NKG2D.

[0356] In other embodiments, the isolated multispecific antibody or its multispecific antigen-binding fragment may comprise: a1) Contains a first antigen-binding site selected from variants of CDR1, CDR2, and CDR3 from Table 15A, or a2) Contains the first antigen binding site of variants of CDR1, CDR2, and CDR3 selected from Table 15B; and b) The second antigen binding site that specifically binds to CD3, CD16, NKp46 or NKG2D.

[0357] In various embodiments, the multispecific antibody comprises two or more VHH domains or two or more single-chain variable fragments (scFv) capable of binding to tumor-associated antigens (TAAs), wherein each of the two or more VHH domains is independently one or more first polypeptides, wherein the one or more first polypeptides include polypeptides selected from Table 14.

[0358] In various embodiments, the multispecific antibody comprises two or more VHH domains or two or more single-chain variable fragments (scFv) capable of binding to a tumor-associated antigen (TAA), wherein each of the two or more VHH domains is independently one or more first polypeptides, wherein the one or more first polypeptides comprises polypeptides having sequences as shown in SEQ ID NO:335 to SEQ ID NO:339.

[0359] In various embodiments, the multispecific antibody comprises two or more VHH domains or two or more single-chain variable fragments (scFv) capable of binding to a tumor-associated antigen (TAA), wherein each of the two or more VHH domains is independently one or more first polypeptides, wherein the one or more first polypeptides comprises a polypeptide having a sequence as shown in SEQ ID NO:335, SEQ ID NO:338 to SEQ ID NO:394.

[0360] Variants of CDR1, CDR2, and CDR3 are discussed herein. In various embodiments, the first antigen-binding site further comprises FWR1, FWR2, FWR3, and FWR4 selected from Table 15B. In various embodiments, the first antigen-binding site further comprises FWR1, FWR2, FWR3, and FWR4 derived from human IgG. In various embodiments, the second antigen-binding site immune-specifically binds to CD3. In various embodiments, the antigen-binding site immune-specifically binds to NKG2D.

[0361] Suitable antigen-binding sites may be in the form of VHH, scFv, Fab, (Fab')2, one or more CDRs, or fusions (optionally tandem sequences linked by a linker) of one or more of VHH, scFv, Fab, (Fab')2, or CDRs. Suitable antigen-binding sites for immune-specific binding of ROR1 include any of the anti-ROR1 antibodies disclosed above or their ROR1 binding fragments. Suitable antigen-binding sites for immune-specific binding of CD3 may be derived from CD3 antibodies disclosed in one or more publications, including but not limited to U.S. Patent No. 8,236,308, U.S. Patent Application Publication Nos. 2010 / 0260668, 2013 / 0018174, 2012 / 0321626, 2013 / 0060011, 2013 / 0058936, 2013 / 0078249, and 2013 / 0058937.

[0362] In some embodiments, the antigen-binding site of the multispecific antibody disclosed herein is linked to a mutated Ig heavy chain constant region. In some aspects, the "kidney-mortar" (KiH) mutation is present in the CH3 domains of both arms (or chains) of the Ig heavy chain constant region, allowing for heterodimerization. This structural feature in the polypeptide arms allows for the assembly of two half-antibodies (e.g., Fc heterodimers; and VH-CH and VL-CL domains). For example, a heteropolymer (including a heterodimer) may comprise a first polypeptide and a second polypeptide, each containing a CH3 domain, wherein the two polypeptides are interposed at an engineered interface within the CH3 domain, and the first polypeptide contains an engineered protrusion ("kidney") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume larger than the original residue; and the second polypeptide contains an engineered cavity ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume smaller than the original residue. In some embodiments, the engineered interface of the heteropolymer comprises at least two pairs of protrusion-cavity mutants. The volume and accessible surface area of ​​each amino acid are described in AA Zamyatnin, Prog. Biophys., Mol. Biol. 24: 107-123, 1972; and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, the lead-in residues used to form the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the lead-in residues used to form the cavity may be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residues used to form the cavity have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "pestle" / protrusion chain and the T366S / L368A / Y407V mutation in the CH3 domain of the "mortar" / cavity chain. Additionally, the KiH configuration can be coupled with further mutations to allow SS disulfide bond connections between the two chains.

[0363] In some embodiments, two or more arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) may contain designs such as another symmetric-asymmetric stereocomplementary design (e.g., HA-TF, ZW1), charge-exchange interaction (DD-KK), charge-stereocomplementary exchange plus additional long-range electrostatic interaction (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic redirection, A107, Duobody, thereby forming a heterodimer / heteromultimer. Further descriptions of these conformations and exemplary mutations / residues can be found, for example, in FrontImmunol. 2016; 7: 394.

[0364] Further embodiments specify that the multispecific antibody may also contain one or more linkers, such as amino acid linkers. In some embodiments, at least one linker is located between the Fc or HSA and at least one antigen-binding site. In some embodiments, at least one linker is located between two antigen-binding sites.

[0365] In some embodiments, the multispecific antibody comprises (1) a "mortar and pestle" IgG Fc domain, (2) a CD3-specific Fab at the N-terminus of the "mortar" chain (or "pestle" chain) of the IgG Fc domain, and (3) two tandemly fused ROR1-specific VHHs at the N-terminus of the "retainer" chain (or "mortar" chain), optionally linked by a linker (VHH-linker-VHH). The two tandemly fused ROR1-specific VHHs herein may be two 2A11s, two 5A1s, or one 2A11 and one 5A1, two 2A11 variants, or two 5A1 variants. 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.

[0366] In some embodiments, the multispecific antibody comprises (1) a "mortar and pestle" IgG Fc domain, (2) a CD3-specific Fab (containing a constant domain and a variable domain of each of the CD3-specific heavy chain and the CD3-specific light chain) at the N-terminus of the "mortar and pestle" chain (or "mortar and pestle" chain) of the IgG Fc domain, (3) a first ROR1-specific VHH fused to the N-terminus of the "mortar and pestle" chain (or the "mortar and pestle" chain respectively), and (4) a second ROR1-specific VHH fused to the C-terminus of the CD3-specific light chain. The first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different and are independently selected from 2A11, 5A1, or variants of 2A11 or 5A1. Exemplary multispecific antibodies of these embodiments include the fusion protein FUSE-394, the amino acid sequence of which is shown in Table 8. In some embodiments, the Fc domain is selected from Table 17A.

[0367] SEQ ID NO: 532 is an exemplary heavy chain resisting CD3 Fab, and SEQ ID NO: 479 is an exemplary light chain resisting CD3 Fab. In various embodiments, the anti-CD3 Fab is constant and is sometimes present on the pestle Fc and sometimes on the mortar Fc.

[0368] In some embodiments, the multispecific antibody comprises (1) a mortar and pestle IgG Fc domain, (2) a CD3-specific Fab fused to the N-terminus of the mortar and pestle chain (or mortar chain) of the IgG Fc domain (containing a constant domain and a variable domain of each of the CD3-specific heavy chain and the CD3-specific light chain), (3) two ROR1-specific VHHs fused in tandem to the N-terminus of the mortar and pestle chain (or mortar 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 tandem ROR1-specific VHHs and the third ROR1-specific VHH may be the same or different, and are each independently selected from 2A11, 5A1, or a variant of 2A11 or a variant of 5A1. In other embodiments, the first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different, and they are independently selected from Table 14. In some embodiments, the Fc domain is selected from Table 17A.

[0369] In some embodiments, the multispecific antibody comprises (1) a "mortar and pestle" IgG Fc domain, (2) a CD3-specific Fab fused to the N-terminus of the "mortar" chain (or "pestle" chain) of the IgG Fc domain, (3) a first ROR1-specific VHH fused to the N-terminus of the "pestle" chain (or "key" chain) of the IgG Fc domain, and (4) a second ROR1-specific VHH fused to the N-terminus of the CD3-specific light chain of the Fab. The first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different, and are independently selected from 2A11, 5A1, or variants of 2A11 or 5A1. In other embodiments, the first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different, and they are independently selected from Table 14. In some embodiments, the Fc domain is selected from Table 17A.

[0370] In some embodiments, the multispecific antibody comprises (1) a mortar and pestle IgG Fc domain, (2) a CD3-specific Fab fused to the N-terminus of the mortar and pestle strand (or mortar strand) of the IgG Fc domain, (3) a first ROR1-specific scFV fused to the N-terminus of the mortar and pestle strand (or mortar strand, respectively) of the IgG Fc domain, and (4) a second ROR1-specific scFV fused to the C-terminus of the mortar and pestle strand (or mortar strand, respectively) of the IgG Fc domain. The first ROR1-specific scFV and the second ROR1-specific scFV may be the same or different. In some embodiments, the Fc domain is selected from Table 17A.

[0371] In some embodiments, the multispecific antibody comprises (1) a mortar and pestle IgG Fc domain, (2) a CD3-specific Fab fused to the N-terminus of the mortar chain (or pestle chain) of the IgG Fc domain, (3) a first ROR1-specific VHH fused to the N-terminus of the pestle chain (or mortar chain) of the IgG Fc domain, and (4) a second ROR1-specific VHH fused to the C-terminus of the CD3-specific light chain of the Fab. The first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different and are independently selected from 2A11, 5A1, or a variant of 2A11 or a variant of 5A1. In other embodiments, the first ROR1-specific VHH and the second ROR1-specific VHH may be the same or different and are independently selected from Table 147. In some embodiments, the Fc domain is selected from Table 17A.

[0372] In various embodiments, the disclosed multispecific antibody (e.g., possessing at least immune specificity for ROR1 and CD3) can bind to ROR1 with an epigenetic binding affinity that typically increases with increasing ROR1 density on the cell surface of the target cell (i.e., corresponding to binding to EC). 50 —The antibody concentration at which 50% of the maximum binding is achieved with certain target cell populations expressing ROR1, which typically tends to decrease. In other words, in various implementations, the binding affinity of a multispecific antibody to cells expressing ROR1 with high ROR1 expression levels can be greater than that to cells with low or zero ROR1 expression levels.

[0373] In various embodiments, a system comprising the following can induce cytotoxicity against ROR1-positive cells (i.e., tumor cells expressing ROR1): a disclosed multispecific antibody (e.g., immune specificity to at least ROR1 and CD3) and a population of CD3-positive immune effector cells, or a CD3-positive fraction of monocytes, or a population of monocytes containing a CD3-positive fraction.

[0374] In some embodiments, multispecific antibodies containing an antigen-binding site that specifically binds to CD3 can bind to CD3-ε on primary human T cells and / or primary cynomolgus monkey T cells. In some embodiments, multispecific antibodies containing an antigen-binding site that specifically binds to CD3 activate the cytotoxicity of primary human CD3+ T cells and / or primary cynomolgus monkey CD3+ T cells. Typically, CD8+ T cells are cytotoxic, while CD4+ T cells primarily “help” CD8+ T cells by secreting cytokines. In some cases, 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 is generally associated with killing target cells via the perforin / granzyme pathway, which activates caspase 3, leading to subsequent apoptosis of the target cells. Other mechanisms of targeted killing include, but are not limited to, activation of target cells expressing Fas, TNF-R, and DR4. TCE directly induces cytotoxicity and can also indirectly induce other targeted killing mechanisms (Sandra Ross et al., PLoSOne, August 24, 2017; 12(8):e0183390).

[0375] In some embodiments, the disclosed multispecific antibody can induce cytotoxicity against ROR1-positive cells (i.e., tumor cells expressing ROR1) in the presence of a population of CD3-positive immune effector cells, CD3-positive fractions of monocytes, or a population of monocytes containing CD3-positive fractions. In some embodiments, when a population of CD3-positive immune effector cells, CD3-positive fractions of monocytes, or a population of monocytes containing CD3-positive fractions are present, the cytotoxicity induced by the disclosed multispecific antibody against ROR1-positive cells with high ROR1 expression levels may be greater than the cytotoxicity induced against ROR1-negative cells or ROR1-positive cells with low ROR1 expression levels.

[0376] Further embodiments specify that, instead of having CD3 specificity or, in addition to CD3 specificity, the multispecific antibody of the present invention may also have specificity against another activating receptor and / or co-stimulatory receptor and / or co-activating receptor commonly expressed on immune cells, in addition to specificity against ROR1. For example, the activating receptor may be expressed on T cells, such that the multispecific antibody can be a T cell connective whose immune specificity binds to the activating receptor on T cells and activates the cytotoxic or cytokine expression properties of T cells. Alternatively, the activating receptor may be expressed on natural killer (NK) cells, such that the multispecific antibody can be an NK cell connective whose immune specificity binds to the activating receptor on NK cells and activates the cytotoxicity of NK cells. Furthermore, the activating receptor may be expressed on various immune cells, such as T cells, NK cells, and dendritic cells (DCs). Therefore, multispecific antibodies can be trifunctional immune cell connectors or trispecific antibodies that bind to two or more activation receptors (such as CD3, CD16, γ9 TCR, δ2 TCR or δ1 TCR, NKp46, CD137 and CD40) to induce immune cell activity, and also bind to ROR1 positive cells to induce immune cell activity against ROR1 positive cells.

[0377] Exemplary activating receptors that can be specifically bound by the antigen-binding sites of the multispecific antibodies disclosed herein include, but are not limited to, CD16, γ9 TCR, δ2 TCR or δ1 TCR, NKp46, CD137, CD40 or NKG2D. Exemplary antigen-binding fragments targeting said activating receptors and / or co-stimulatory receptors and / or co-activating receptors include scFv, VH, VL, VHH, Fab, etc., which are described in various publications, including U.S. Patent No. 9,035,026 and Gauthier et al., 2019, Cell 177, 1701-1713. Further exemplary antigen-binding antibodies and / or fragments targeting said activating receptors and / or co-stimulatory receptors and / or co-activating receptors include those antigen-binding antibodies and / or fragments shown in Table 18. Among the listed antigen-binding antibodies, their VHH or Fab regions can be used as antigen-binding sites for the multispecific antibodies disclosed herein.

[0378] Another embodiment provides a ROR1-specific antibody-drug conjugate (ADC) comprising an anti-ROR1 antibody or its ROR1-binding fragment disclosed herein, and a pharmaceutical unit conjugated to or bound to the anti-ROR1 antibody or its ROR1-binding fragment. Optionally, a linker unit is located between the anti-ROR1 antibody or its ROR1-binding fragment and the pharmaceutical unit, and the linker unit may be an amino acid linker or a chemical moiety linker. Exemplary pharmaceutical units may be cytotoxic agents, such as antitumor drugs or chemotherapeutic agents, including small molecules and siRNA.

[0379] In other embodiments, the multispecific antibody, multispecific antigen-binding fragment, or composition comprising thereof may also involve additional therapeutic agents / therapies as needed for treating a specific condition. Preferably, the multispecific antibody or its multispecific antigen-binding fragment and the additional therapeutic agent have complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent / therapy is a chemotherapeutic agent or radiotherapy. The combined administration of the disclosed multispecific antibody or its multispecific antigen-binding fragment and the additional therapeutic agent can be performed simultaneously, separately, or sequentially in any order. For simultaneous administration, these therapeutic agents can be administered as a composition or as separate compositions as needed.

[0380] The dosage of a multispecific antibody, multispecific antigen-binding fragment, or composition comprising the thereof depends on the desired effect, duration of treatment, and route of administration. For example, therapeutically effective amounts of the multispecific antibodies disclosed herein include one or more doses, one of which is in the range of: about 10 mg to 50 mg, 50 mg to 100 mg, 100 mg to 150 mg, 150 mg to 200 mg, 100 mg to 200 mg, 200 mg to 300 mg, 300 mg to 400 mg, 400 mg to 500 mg, 500 mg to 600 mg, 600 mg to 700 mg, 700 mg to 800 mg, 800 mg to 900 mg, 900 mg to 1000 mg, 1000 mg to 1100 mg, 1100 mg to 1200 mg, and 1200 mg to 1300 mg. 1300mg to 1400mg, 1400mg to 1500mg, 1500mg to 1600mg, 1600mg to 1700mg, 1700mg to 1800mg, 1800mg to 1900mg, 1900mg to 2000mg, 2000mg to 2100mg, 2100mg to 2200mg, 2200mg to 2300mg, 2300mg to 2400mg, 2400mg to 2500mg, 2500mg to 2600mg, 2600mg to 2700mg, 2700mg to 2800mg, 2800mg to 2900mg, or 2900mg to 3000mg.In another embodiment, the therapeutically effective amount of the multispecific antibody disclosed herein comprises one or more doses, wherein one dose is in the following ranges: 0.001 mg / kg to 0.005 mg / kg, 0.005 mg / kg to 0.01 mg / kg, 0.01 mg / kg to 0.02 mg / kg, 0.02 mg / kg to 0.04 mg / kg, 0.04 mg / kg to 0.06 mg / kg, 0.06 mg / kg to 0.08 mg / kg, 0.08 mg / kg to 1 mg / kg, 1 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, 15 mg / kg to 2 mg / kg. 0 mg / kg, 20 mg / kg to 25 mg / kg, 25 mg / kg to 30 mg / kg, 30 mg / kg to 35 mg / kg, 35 mg / kg to 40 mg / kg, 40 mg / kg to 45 mg / kg, 45 mg / kg to 50 mg / kg, 10 mg / kg to 50 mg / kg, 50 mg / kg to 100 mg / kg, 100 mg / kg to 150 mg / kg, 150 mg / kg to 200 mg / kg, 100 mg / kg to 200 mg / kg, 200 mg / kg to 300 mg / kg, 300 mg / kg to 400 mg / kg, 400 mg / kg to 500 mg / kg, 5 00mg / kg to 600mg / kg, 600mg / kg to 700mg / kg, 700mg / kg to 800mg / kg, 800mg / kg to 900mg / kg, 900mg / kg to 1000mg / kg, 1000mg / kg to 1100mg / kg, 1100mg / kg to 1200mg / kg, 1200mg / kg to 1300mg / kg, 1300mg / kg to 1400mg / kg, 1400mg / kg to 1500mg / kg, 1500mg / kg to 1600mg / kg, 1600mg / kg to 1700mg / kg, 1700mg / kg to 1800mg / kg g / kg, 1800mg / kg to 1900mg / kg, 1900mg / kg to 2000mg / kg, 2000mg / kg to 2100mg / kg, 2100mg / kg to 2200mg / kg, 2200mg / kg to 2300mg / kg, 2300mg / kg to 2400mg / kg, 2400mg / kg to 2500mg / kg, 2500mg / kg to 2600mg / kg, 2600mg / kg to 2700mg / kg, 2700mg / kg to 2800mg / kg, 2800mg / kg to 2900mg / kg, or 2900mg / kg to 3000mg / kg.In some implementations, a therapeutically effective dose includes two, three, or more doses administered daily, weekly, bi-weekly, monthly, quarterly, or annually; or treatment continues until the disease is successfully treated or the subject no longer experiences symptoms or signs of the disease when assessments show improvement in the severity of the disease compared to the severity prior to the last dose.

[0381] Table 8. Amino acid sequences of exemplary fusion proteins. Peptide 1 contains the "potassium" of KiH Fc; peptide 2 contains KiH Fc. The "pestle"; and polypeptide 3 contains a light chain. VHH can be contained in polypeptide 1, polypeptide 2, and / or polypeptide 3, and there are connections between them. Head. In this table, the anti-CD3 domain is located at the N-terminus of the pestle Fc or mortar Fc. .

[0382] Table 9. Which polynucleotide sequences encoding fusion proteins correspond to those in Table 8? .

[0383] Table 10. Amino acid sequence of an exemplary VHH .

[0384] Table 11. The polynucleotide sequences encoding VHH are shown in Table 10. .

[0385] Table 12. Expression yields of some fusion proteins as shown in the examples. .

[0386] Table 13. Melting temperature (Tm) of exemplary fusion proteins .

[0387] IL-15 variants and IL-15 fusion protein Various embodiments of the present invention provide IL-15 variants.

[0388] Various implementations provide a variant of interleukin-15 (IL-15) that comprises the sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKE FLQSX 103 VHIVX 108 MFIX 112 TS, in which X1 can be any amino acid. X4 can be any amino acid. X 22 It can be any amino acid. X 30 It can be any amino acid. X 41 It can be any amino acid. X 45 It can be any amino acid. X49 It is any amino acid except V. X 53 It is any amino acid except E. X 64 It can be any amino acid. X 67 It can be any amino acid. X 68 It can be any amino acid. X 79 It can be any amino acid. X 93 It can be any amino acid. X 103 It can be any amino acid. X 108 It is any amino acid, and X 112 It is any amino acid; (SEQ ID NO:533).

[0389] In various embodiments of the IL-15 variant, X1 is N (SEQ ID NO:534), X4 is N (SEQ ID NO:535), X 22 It is D (SEQ ID NO:536), X 30 It is D (SEQ ID NO:537), X 41 It is K (SEQ ID NO:538), X 45 It is L (SEQ ID NO: 539), X 64 It is E (SEQ ID NO:540), X 67 It is I (SEQ ID NO:541), X 68 It is I (SEQ ID NO:542), X 79 It is N (SEQ ID NO:543), X 93 It is E (SEQ ID NO:544), X 103 It is F (SEQ ID NO:545), X 108 It is Q (SEQ ID NO:546), or X 112 It is N (SEQ ID NO: 547), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of them.

[0390] In various embodiments of the IL-15 variant, X1 is N (SEQ ID NO:534), X4 is N (SEQ ID NO:535), X 22 It is D (SEQ ID NO:536), X 30 It is D (SEQ ID NO:537), X 41 It is K (SEQ ID NO:538), X 45 It is L (SEQ ID NO: 539), X 49 It is R or K (SEQ ID NO:548), X 53 It is G, K, I or A (SEQ ID NO:549), X 64 It is E (SEQ ID NO:540), X 67 It is I (SEQ ID NO:541), X 68 It is I (SEQ ID NO:542), X 79 It is N (SEQ ID NO:543), X 93 It is E (SEQ ID NO:544), X 103 It is F (SEQ ID NO:545), X 108 It is Q (SEQ ID NO:546), or X 112 It is N (SEQ ID NO: 547), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of them.

[0391] In various embodiments of the IL-15 variant, X1 is N or G (SEQ ID NO:550), X4 is N, K, or L (SEQ ID NO:551), X 22 It is D or A (SEQ ID NO:552), X 30 It is D or N (SEQ ID NO:553), X 41 It is K or Q (SEQ ID NO:554), X 45 It is L or S (SEQ ID NO:555), X 49 It is R or K (SEQ ID NO:548), X 53 It is G, K, I or A (SEQ ID NO:549), X 64 It is E or Q (SEQ ID NO:556), X 67 It is I or T (SEQ ID NO:557), X 68It is I or S (SEQ ID NO:558), X 79 It is N or Y (SEQ ID NO:559), X 93 It is E or A (SEQ ID NO:560), X 103 It is F or L (SEQ ID NO:561), X 108 It is Q or T (SEQ ID NO:562), or X 112 It is N or R (SEQ ID NO: 563), or any combination thereof. In various embodiments, any combination thereof is a combination of any two of them. In various embodiments, any combination thereof is a combination of any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of them.

[0392] In various implementations of IL-15 variants, X 49 It is R or K, and X 53 It is G, K, I or A; (SEQ ID NO:564).

[0393] In various implementations of IL-15 variants, X 30 It is N (SEQ ID NO:565).

[0394] In various implementations of IL-15 variants, X 64 It is Q (SEQ ID NO:566).

[0395] In various implementations of the IL-15 variant, X1 is G (SEQ ID NO:567).

[0396] In various implementations, the IL-15 variants are selected from those in Table 1A or Table 3A.

[0397] In various embodiments, the IL-15 variants are not those described in U.S. Patent Application Publication No. US 2019 / 0263877, which are incorporated herein by reference.

[0398] This document also provides fusion proteins comprising an IL-15 variant and a scaffold peptide. Various embodiments of the invention provide a fusion protein comprising an IL-15 variant as described herein and a scaffold peptide. In various embodiments, the fusion protein comprises two or more IL-15 variants as described herein and a scaffold peptide. In various embodiments, the two or more IL-15 variants may be three, four, five, or six IL-15 variants. In various embodiments, the two or more IL-15 variants may be up to eight IL-15 variants.

[0399] In various implementations, the fusion protein also includes one or more immune checkpoint targeting fragments.

[0400] In various embodiments, immune checkpoints include, but are not limited to, PD-1, PD-L1, CTLA-4, and LAG-3. In various embodiments, one or more immune checkpoint targeting fragments include immune checkpoint targeting fragments of known antibodies. Examples of anti-PD1 fragments include fragments from pembrolizumab, nivolumab, pildilizumab, AMP-224, AMP-514, spartazumab, cimiprizumab, penamprizumab (AK105), palolizumab (BCD-100), ebbenlimumab (BI754091), toripalimab (JS001), lipusubibumab (LZM009), rivalimab (MGA012), Sym021, dostalimumab (TSR-042), terpolizumab (MGD013), candunizumab (AK104), vodalizumab (XmAb20717), tislelizumab, or PF-06801591 (e.g., Fab, Fv). Other examples of anti-PD1 fragments include fragments (e.g., Fab, Fv) from vopalimumab, camrelizumab, sintilimab, AMP-224, AMP-514, and acrixolimab. Examples of anti-PD-L1 antibodies include, but are not limited to, gorevolimab (BGB-A333), cochilimumab (CK-301), FAZ053, envorimab (KN035), MDX-1105, betifisolimab (MSB2311), adebenone (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824. Thus, for example, fusion proteins comprise, in addition to, anti-PD1 fragments (e.g., Fab, Fv) from anti-PD1 antibodies and IL-15 variants, and optionally, linkers.

[0401] In various embodiments, the IL-15 variant is fused to the C-terminus of the scaffold peptide. In other embodiments, the IL-15 variant is fused to the N-terminus of the scaffold peptide.

[0402] In some other embodiments, the scaffold peptide is an antibody, and the IL-15 variant is fused to either the C-terminus or N-terminus of the heavy or light chain, or to C... H 2. Merge either the structural domain or the hinge region (e.g., located in C) H 2. Structural Domains and C H 1. Between structural domains.

[0403] 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.

[0404] In various implementations, the antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, pildizumab, AMP-224, AMP-514, spartazumab, cimiprizumab, penampilimab (AK105), palolizumab (BCD-100), ebbenlimumab (BI 754091), toripalimab (JS001), lipusubibumab (LZM009), rivalimab (MGA012), Sym021, dostalimumab (TSR-042), terpolilimumab (MGD013), candunilimumab (AK104), vordalilimumab (XmAb20717), tislelizumab, PF-06801591, and pluripotent cytotoxic T lymphocytes expressing anti-PD1 antibodies (PIK-PD-1). Examples of anti-PD-L1 antibodies include, but are not limited to, gliovelimab (BGB-A333), cosivelimab (CK-301), FAZ053, envorimab (KN035), MDX-1105, betifisolimab (MSB2311), adebenone (SHR-1316), atezolizumab, averumab, durvalumab, BMS-936559, CK-301, and M7824.

[0405] In various embodiments, the scaffold peptide is an Fc region or a fragment thereof. In various embodiments, the scaffold peptide is an Fc region or a fragment thereof, and the scaffold peptide does not contain Fab.

[0406] In various embodiments, the two arms (or chains) of the constant region of the immunoglobulin heavy chain (e.g., an Fc polypeptide) can undergo heterodimerization by generating a "mortar and pestle" (KiH) mutation in the CH3 domain. This structural feature in the polypeptide arms allows the assembly of two half-antibodies (e.g., an Fc heterodimer; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) may comprise two polypeptides, each containing a CH3 domain, wherein the two polypeptides interlock at an engineered interface within the CH3 domain, and one polypeptide contains an engineered protrusion ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume larger than the original residue; and the other polypeptide contains an engineered cavity ("mortar") at the interface, wherein at least one contact residue is replaced by an introduction residue with a side chain volume smaller than the original residue. In some embodiments, the engineered interface of the heteromultimer comprises at least two pairs of protrusion-cavity mutants. The volume and accessible surface area of ​​each amino acid are described in AA Zamyatnin, Prog. Biophys., Mol. Biol. 24: 107-123, 1972; and C. Chothia, J. Mol. Biol. 105: 1-14, 1975. For example, the lead-in residues used to form the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residues used to form the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the lead-in residues used to form the cavity may be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residues used to form the cavity have large side chain volumes, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "groove" / protrusion chain and the T366S / L368A / Y407V mutation in the CH3 domain of the "mortar" / cavity chain. Additionally, the KiH conformation can be coupled with further mutations to allow SS disulfide bond linkage between the two chains. In this conformation, the protein / peptide is a heterodimer of the KiH conformation, with IL-18 (or a fragment thereof, a variant thereof, or a fragment of a variant thereof) linked to one and only one of two (or more) immunoglobulin heavy chain constant regions / chains (i.e., groove or mortar).

[0407] Therefore, in some implementations, the Fc region is the mortar and pestle (KiH) Fc.

[0408] In various embodiments, the IL-15 variant is fused with the pestle of the KiH Fc. In various embodiments, the IL-15 variant is fused with the mortar of the KiH Fc.

[0409] In various embodiments, the Fc region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1. In various embodiments, the Fc region can be human IgG1, IgG2, or IgG4.

[0410] In some embodiments, two or more arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) may contain designs such as another symmetric-asymmetric stereocomplementary design (e.g., HA-TF, ZW1), charge-exchange interaction (DD-KK), charge-stereocomplementary exchange plus additional long-range electrostatic interaction (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic redirection, A107, Duobody, thereby forming a heterodimer / heteromultimer. Further descriptions of these conformations and exemplary mutations / residues can be found in FrontImmunol. 2016; 7: 394.

[0411] In various embodiments, the scaffold peptide of the fusion protein includes globular proteins, human serum albumin (HSA), β2 microglobulin, transferrin, fragment antigen-binding domains (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, or fragments thereof.

[0412] In various embodiments, one or more peptides may be inserted between an antibody and an IL-15 variant. In various embodiments, the peptide may be inserted into or conjugated at the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the antibody. In various embodiments, the peptide comprises a peptide linker conjugated to the IL-15 variant and the antibody.

[0413] In various embodiments, one or more peptides may be inserted between the IL-15 variant and the Fc region of the IL-15 fusion protein. In various embodiments, the peptide may be inserted or conjugated at the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the Fc region. In various embodiments, the peptide includes a peptide linker conjugating the IL-5 variant and the Fc region. In these embodiments, the fusion protein does not include the Fab region of the antibody.

[0414] Antibodies that can fuse with IL-15 may 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 antibody may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). Preferably, the antibody is a human antibody or a humanized antibody.

[0415] In various embodiments, the IL-15 fusion protein is the IL-15 fusion protein listed in Table 1A. That is, an IL-15 fusion protein comprising peptide 1, peptide 2, and peptide 3.

[0416] In various implementations, the IL-15 fusion protein includes the IL-15 variants listed in Table 1A or Table 3A.

[0417] In various embodiments, the IL-15 fusion protein comprises peptides 1, 2, and 3 listed in Table 1A, differing only in that the linker in peptide 2 is a different peptide linker. For example, this linker may be a flexible linker, typically about 10 to 25 amino acids in length. Other examples of such linkers include, but are not limited to, two-amino acid dimers, three-amino acid trimers, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 235), (GGGGX). λ (SEQ ID NO:236))n and (X λ GGGG (SEQ ID NO:317))n, where X λ It is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, so that the length of the amino acid linker is 25 amino acids or less.

[0418] This article provides methods for generating IL-15 variants and IL-15 fusion proteins.

[0419] Various embodiments provide a polynucleotide that encodes the IL-15 variant of the present invention described herein. For example, the nucleic acid sequence may encode an IL-15 variant or an IL-15 fusion protein in the 5' to 3' orientation.

[0420] Various embodiments provide a polynucleotide that encodes the IL-15 fusion protein of the present invention as described herein. For example, the nucleic acid sequence may encode an IL-15 variant in the 5' to 3' orientation.

[0421] Therefore, the polynucleotides encoding the IL-15 fusion protein of the present invention include polynucleotides 1, 2, and 3 listed in Table 2A. In other embodiments, the polynucleotides encoding the IL-15 fusion protein of the present invention include polynucleotides 1, 2, and 3 listed in Table 2A, except that the nucleotides encoding the adapter encode different adapters.

[0422] For example, the linker can be a flexible linker, typically about 10 to 25 amino acids in length. Other examples of such linkers include, but are not limited to, dimers of two amino acids, trimers of three amino acids, or peptides selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:235), (GGGGX). λ (SEQ ID NO:236))n and (X λ GGGG(SEQ ID NO:317))n, where X λ It is Q, A, E, or S, and n is an integer from 1 to 5 or greater than 5. In some embodiments, the amino acid linker has an amino acid sequence (GGGGS (SEQ ID NO:237))n, where n is an integer from 1 to 5, so that the length of the amino acid linker is 25 amino acids or less. Therefore, the nucleotide encoding the linker in polynucleotide 2 will instead encode one of these aforementioned linkers.

[0423] Exemplary polynucleotides are shown in Table 2A.

[0424] Various embodiments also provide an expression vector comprising any of the polynucleotides described herein. Therefore, polypeptides encoding IL-15 variants or IL-15 fusion proteins can also be integrated into reproducible expression vectors. Thus, vectors encoding fusion proteins are also provided that can express the fusion protein, for example, in a bacterial host, a intended recipient, or both.

[0425] Another embodiment provides cells transformed or transfected with one or more nucleic acid molecules encoding an IL-15 variant or an IL-15 fusion protein. These cells may be prokaryotic cells. Alternatively, the cells may be eukaryotic cells, preferably mammalian cells, and more preferably human cells.

[0426] In various implementations, the cell is a mammalian cell. Available mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells or HEK-293 cells.

[0427] In various implementations, the cell is a bacterial cell or a yeast cell.

[0428] Various implementations provide a method for generating an IL-15 variant, the method comprising: culturing any of the cells described herein in a cell culture medium to allow the generation of an IL-15 variant, and optionally secreting it into the cell culture medium.

[0429] In various embodiments, the method further includes isolating the IL-15 variant from the cell or from the cell culture medium. In various embodiments, the method further includes purifying the IL-15 variant.

[0430] Various embodiments provide a method for producing an IL-15 fusion protein, the method comprising: culturing any of the cells described herein in a cell culture medium to allow the production of the IL-15 fusion protein, and optionally secreting it into the cell culture medium.

[0431] In various embodiments, the method further includes isolating the IL-15 fusion protein from the cell or from the cell culture medium. In various embodiments, the method further includes purifying the IL-15 fusion protein.

[0432] In some implementations, the Chinese hamster ovary (CHO) expression system is used to generate the IL-15 variant or fusion protein by means of the following steps: (1) cell resuscitation, in which frozen CHO cells can be resuscitated under a water bath at 37°C; (2) cell passage culture, in which the cells can be passaged to adjust the cell density to 6 × 10⁶ cells / year. 6 (3) Transfection and expression: use solution 1 (where the plasmid is diluted with diluent) and solution 2 (where the transfection reagent is diluted with diluent), then mix solution 1, solution 2 and CHO cells, and then incubate the mixture in a shaker at 32°C for 12 to 14 days for expression, and collect the culture supernatant after centrifugation.

[0433] In some embodiments, a purification process is performed after the fusion protein is expressed. In some embodiments, the purification method includes the following steps: (1) washing the column with binding buffer (10 volumes) at a flow rate of 1 mL / min; (2) loading a sample containing the fusion protein at a flow rate of 1 mL / min; (3) washing the column with 10 volumes of PBS buffer at a flow rate of 1 mL / min; (4) eluting the protein from the column with 40 mM sodium citrate (pH 3.4); optionally, the eluted sample may be collected in a tube (1 mL / min) and the optical density (OD) may be measured at 280 nm using NanoDrop; and (5) performing dialysis, for example, overnight dialysis with PBS buffer in a dialysis bag.

[0434] Various embodiments of the present invention provide a method for activating and promoting the differentiation or expansion of T cells, B cells or natural killer (NK) cells, the method comprising administering to a subject in need an IL-15 variant of the present invention as described herein.

[0435] Various embodiments of the present invention provide a method for activating and promoting the differentiation or expansion of T cells, B cells or natural killer (NK) cells, the method comprising administering the IL-15 fusion protein of the present invention, as described herein, to a subject in need.

[0436] In various implementation schemes, the subject had cancer.

[0437] Various embodiments of the present invention provide a method for alleviating, suppressing or treating a disease or symptom in a subject in need, the method comprising administering to the subject in need an IL-15 variant of the present invention as described herein.

[0438] Various embodiments of the present invention provide a method for alleviating, suppressing or treating a disease or symptom in a subject in need, the method comprising administering to the subject in need the IL-15 fusion protein of the present invention as described herein.

[0439] In various implementation schemes, the disease or symptom is cancer.

[0440] Pharmaceutical Composition In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of a fusion protein. "Pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and desirable, and includes excipients acceptable for both veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous.

[0441] In some embodiments, the compounds of the present invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. As used herein, the terms "pharmaceutically acceptable salts, esters, amides, and prodrugs" refer to those carboxylates, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention that are suitable for use in contact with a patient's tissues within a reasonable medical judgment without undue toxicity, irritation, allergic reactions, etc., in accordance with a reasonable benefit / risk ratio, and effectively achieve the intended use of the compounds of the present invention. The term "salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds of the present invention. These salts may be prepared in situ during the final separation and purification of the compounds, or by reacting the purified compound in its free base form alone with a suitable organic or inorganic acid and separating the resulting salt. These salts may include alkali metal and alkaline earth metal-based cations, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (see, for example, Berge SM et al., (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0442] The term "pharmaceutically acceptable ester" refers to the relatively non-toxic esterification products of the compounds of this invention. These esters can be prepared in situ during the final separation and purification of the compounds, or by reacting the purified compound, in its free acid or hydroxyl form, alone with a suitable esterifying agent. Carboxylic acids can be converted to esters by treatment with alcohols in the presence of a catalyst. The term also includes lower hydrocarbon groups capable of being solvated under physiological conditions, such as alkyl esters, methyl esters, ethyl esters, and propyl esters.

[0443] As used in this article, "pharmaceutically acceptable salt or prodrug" means that such salt or prodrug is suitable for use in contact with the tissues of a subject within a reasonable medical judgment without excessive toxicity, irritation, allergic reactions, etc., meets a reasonable benefit / risk ratio, and can effectively achieve its intended use.

[0444] The term "prodrug" refers to a compound that is rapidly converted in vivo to produce one or more functionally active peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein. A comprehensive discussion is provided in the following references: T. Higachi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Volume 14 of the American Chemical Society Symposium Series, and “Bioreversible Carriers in: Drug Design,” edited by Edward B. Roche, published by the American Pharmaceutical Association and Pergamon Press in 1987, both of which are incorporated herein by reference. As used herein, a prodrug is a compound that is converted in vivo, either metabolically or otherwise, to its biologically, pharmaceutically, or therapeutically active form. Prodrugs of one or more peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein, may be engineered to alter the metabolic stability or transport characteristics of one or more peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein, to mask side effects or toxicities, thereby improving the flavor of the compound or altering other characteristics or properties of the compound. With knowledge of pharmacokinetic processes and drug metabolism in vivo, those skilled in the art can typically design prodrugs from one or more peptides, or their mutants, variants, analogs, or derivatives, as disclosed herein, once the pharmaceutically active form is obtained (see, for example, Nogrady (1985), “Medicinal Chemistry: A Biochemical Approach,” Oxford University Press, New York, pp. 388–392). For instance, routine procedures for selecting and preparing suitable prodrug derivatives are described in, for example, H. Bundgaard’s “Design of Prodrugs,” Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl, and glycerol esters of the corresponding acids.

[0445] In various embodiments, the pharmaceutical composition according to the invention can be formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, mucosal, transdermal, or parenteral administration.

[0446] Transdermal application can be accomplished using topical creams or ointments or transdermal patches.

[0447] "Parenteral" refers to routes of administration typically associated with injection, including intraorbital, infusion, intra-arterial, intra-bursal, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subbursal, subcutaneous, transmucosal, or transtracheal routes. Through the parenteral route, the composition may be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder.

[0448] via the enteral route, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles, thereby allowing controlled release. via the parenteral route, the composition may be in the form of a solution or suspension for infusion or injection.

[0449] Pharmaceutical compositions based on the compounds of this invention can be formulated for the treatment of skin and mucous membranes via a local route, and are in the form of ointments, creams, emulsions, powders, dipping 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, thereby allowing controlled release. These local route compositions can be in anhydrous or aqueous forms, depending on the clinical indication.

[0450] They can be taken as eye drops via the ocular route.

[0451] The pharmaceutical compositions according to the invention may also contain any pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium that participates in carrying or transporting the compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, a 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" because it must be compatible with the other components of the formulation. The carrier must also be suitable for contact with any tissue or organ it may come into contact with, meaning it must not carry a risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that do not excessively outweigh its therapeutic benefits.

[0452] The pharmaceutical compositions according to the invention can also be encapsulated, tableted, or prepared as emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. Carriers may also include sustained-release materials (such as glyceryl monostearate or glyceryl distearate) which may be used alone or in combination with waxes.

[0453] The pharmaceutical formulation is prepared according to standard pharmaceutical techniques, including grinding, mixing, granulation, and, where necessary, compression into tablet form; or grinding, mixing, and filling into hard gelatin capsules. When a liquid carrier is used, the formulation will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. This liquid formulation can be administered directly orally or filled into soft gelatin capsules.

[0454] The pharmaceutical compositions according to the invention can be delivered in therapeutically effective amounts. A precise therapeutically effective amount is the amount of composition that produces the most effective therapeutic outcome in a given subject. This amount will vary depending on a variety of factors, including, but not limited to: the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art of clinical and pharmacology will be able to determine the therapeutically effective amount through routine experiments, such as by monitoring the subject's response to the compound and adjusting the dose accordingly. For further guidance, see “Remington: The Science and Practice of Pharmacy” (edited by Gennaro, 20th edition, Williams & Wilkins, Pennsylvania, USA, 2000).

[0455] Reagent test kit The present invention also relates to a kit for treating various diseases. The kit is a combination of materials or components comprising at least one composition of the present invention. Therefore, in some embodiments, the kit contains a composition comprising the fusion protein described above.

[0456] The exact properties of the components configured in the kit of the present invention depend on their intended purpose. For example, some embodiments are configured for the purpose of activating, promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells. Some embodiments are configured for the purpose of treating cancer. In one embodiment, the kit is specifically configured for the purpose of treating mammalian subjects. In another embodiment, the kit is specifically configured for the purpose of treating human subjects. In a further embodiment, the kit is configured for veterinary applications to treat subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0457] The kit may include instructions for use. These instructions typically include a detailed description of the techniques used when using the kit components to achieve desired results, such as treating a disease (including cancer) by activating, promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells. Optionally, the kit may also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring instruments, bandage materials, or other useful instruments readily identifiable by those skilled in the art.

[0458] Materials or components assembled in this kit can be stored and provided to practitioners in any convenient and suitable manner to maintain their operability and usability. For example, these components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room temperature, refrigerated, or frozen temperatures. These components are typically contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the present invention. Packaging materials are prepared by well-known methods, preferably providing a sterile and uncontaminated environment. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of containing individual kit components. Thus, for example, packaging can be a glass vial for containing a suitable amount of the compositions of the present invention containing an IL-15 variant or fusion protein. Packaging materials typically have an external label indicating the contents and / or purpose of the kit and / or its components.

[0459] Example The following examples are intended to illustrate the claimed invention more clearly and should not be construed as limiting the scope of the invention. The specific materials mentioned are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without inventive step and without departing from the scope of the invention.

[0460] Example IL15 / IL18 variant examples Example 1 As shown in Figure 2, we evaluated the effects of an exemplary anti-PD1-pro-IL-18 antibody, one of three exemplary anti-PD1-mutant-IL-15 antibodies, or a combination of an anti-PD1-pro-IL-18 antibody with each of the three mutant IL-15 antibody fusions on IFNγ release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (Pelicluster CD3, which is capable of crosslinking with CD3 and activating T cells without FcγR binding) for up to 72 hours. For anti-PD1-pro-IL-18, the exemplary assay was Fuse694, which incorporates a pro-IL-18 (a granzyme B-cleavable IL18mutAS) into the C-terminal mortise or tenon of the IgG1-LALA version of nivolumab (Fuse691). For anti-PD1 mutant-IL-15, the three assays were Fuse765 (… Figure 2A Hollow triangle, dashed line), Fuse773 ( Figure 2B (Hollow triangle, dashed line) and Fuse774 ( Figure 2C (Hollow triangle, dashed line) These are obtained by incorporating an IL-15m4, IL15m9, or IL15m10 into the C-end pestle or mortar of Fuse691, respectively. Figures 2A to 2C It is a nonlinear xy graph showing the relationship between the concentration of the test sample and the release of IFNγ.

[0461] In each relational plot, (1) a chicken egg lysozyme (HEL) specific antibody (i.e., anti-HEL) (gray circle) was used as a human IgG1 isotype control and correlated with background IFNγ release. Fuse691 (hollow square, dashed line) was observed to induce mild IFNγ release in this system, equivalent to a maximum of approximately 1200 pg / ml. Using Fuse694, Fuse765, Fuse773, and Fuse774 to target PD1 individually with pro-IL-18 or any of the three IL-15 mutants resulted in only modest increases in the observed maximum IFNγ release relative to Fuse691, with values ​​of approximately 1900 pg / ml, 3000 pg / ml, 2800 pg / ml, and 1700 pg / ml, respectively. In contrast, the combinations of Fuse765 plus Fuse694 (black triangle), Fuse773 plus Fuse694 (black triangle), and Fuse774 plus Fuse694 (black triangle) resulted in maximum observed IFNγ releases of approximately 12,000 pg / ml, 11,500 pg / ml, and 7,400 pg / ml, respectively. These values ​​are 4 to 6 times higher than any one of the individual anti-PD1-pro-IL-18 (Fuse694) and / or anti-PD1-mutant-IL15 variants (Fuse765, Fuse773, and Fuse774), indicating that the combined effects of targeting both IL-15 and IL-18 to PD1 in this system are synergistic. Interestingly, although EC50-IFNγ releases for the single-cytokine fusions of Fuse691 and anti-PD1 appear to cluster within a 2 to 3-fold range.

[0462] The sum of EC50, Emax, and AUC (area under the curve) values ​​is expressed as follows: Figure 2D middle.

[0463] Example 2 like Figure 3As shown, we evaluated the effects of exemplary anti-PD1-pro-IL-18 antibody, exemplary anti-PD1-mutant-IL-15 antibody, and anti-PD1 antibody incorporating both the aforementioned exemplary pro-IL-18 and mutant IL-15 on IFNγ release mediated by purified T cells exposed to a suboptimal dose of anti-CD3 (PeliCluster CD3, which can crosslink with CD3 and activate T cells without FcγR binding) for up to 72 hours. For anti-PD1-pro-IL-18, the exemplary assay is Fuse694 (black square). For anti-PD1 mutant-IL-15, the exemplary assay is Fuse696 (black rhombus), which is obtained by incorporating one IL-15 m2 into the C-terminal pestle or mortar of an anti-PD1 human IgG1 LALA antibody (i.e., Fuse691) (hollow square, dashed line). Finally, Fuse697 (black triangle) is obtained by incorporating a pro-IL18 into the C-terminal pestle of Fuse691 and an IL-15m2 into the C-terminal mortise. Figure 3This is a non-linear xy plot showing the relationship between test sample concentration and IFNγ release. Human IgG1 antibody (i.e., anti-HEL) (gray circle) was used as an isotype control and correlated with background IFNγ release. Fuse691 (hollow square, dashed line) was observed to induce mild IFNγ release in this system, equivalent to a maximum of approximately 830 pg / ml. Targeting PD1 with pro-IL-18 alone using Fuse694 resulted in only a modest increase in the observed maximum IFNγ release relative to Fuse691, at approximately 2,000 pg / ml. Targeting PD1 with IL-15m2 alone using Fuse696 resulted in a more significant increase in the observed maximum IFNγ release relative to Fuse691, at approximately 9,000 pg / ml. The maximum observed IFNγ release induced by Fuse697 was approximately 21,700 pg / ml, which is about 11 times the maximum IFNγ release observed by Fuse694 and 2.5 times the maximum IFNγ release observed by Fuse696. Importantly, the maximum observed IFNγ release of Fuse697 is equivalent to greater than the sum of the maximum IFNγ release values ​​associated with Fuse694 plus Fuse696, suggesting that IL-15 and IL-18 act synergistically when co-incorporated into anti-PD1. Interestingly, unlike the combination of anti-PD1-pro-IL-18 and anti-PD1-mutant-IL15 relative to the EC50-IFNγ release of the single-cytokine fusion of Fuse691 and anti-PD1, the EC50-IFNγ release of Fuse697 was significantly shifted to the left (more effective), at least 30 times that of Fuse691, Fuse694, or Fuse696. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below the xy graph.

[0464] Example 3 As shown in Figure 4, we evaluated the ability of NKG2D xROR1 bispecific antibodies (bsAbs) with or without exemplary pro-IL18 and IL15 mutants to induce NK cell-mediated tumor cell killing and IFNγ release. The NKG2D xROR1 bsAb was human IgG1 LALA (the parental bsAb without cytokine incorporation was Fuse926), obtained by incorporating pro-IL18 (a granzyme B-cleavable IL18mutAV) at the N-terminal lamina and IL15m9-1 (Fuse916) at the C-terminal lamina. NK cells were expanded and enriched from human PBMCs (normal donors) and used as effector cells. ROR1+ tumor cells were MDA-MB-231 (MDA-GL) stably expressing firefly luciferase and Egfp. NK cells were mixed with MDA-GL at a 5:1 effector-to-target ratio (E:T) for 48 hours. Tumor cell killing was assessed by luciferase activity, and IFNγ release in culture supernatant was measured by ELISA. Figure 4A and Figure 4B These are non-linear x-y plots showing the relationship between tumor cell killing and assay concentration, and between IFNγ release and assay concentration. Compared to Fuse926 (black square), Fuse916 (black triangle) induced approximately 50% more maximum cell killing and approximately 7-fold more maximum IFNγ release. The potency of Fuse916 (defined as EC50-kill or EC50-IFNγ release) was also improved compared to Fuse916, with approximately 5-fold and >10-fold increases in tumor cell killing and IFNγ release, respectively. Anti-HEL Ab served as a negative isotype control, and Fuse923 (black rhombus), i.e., γ9δ2-TCR x ROR1 bsAb, incorporated pro-IL18 and IL15m9-1 along the same orientation as an assay for incorporating non-targeted cytokines. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each x-y plot.

[0465] Example 4 As shown in Figure 5, we evaluated the effect of targeting NKG2D with exemplary IL-15 variants and exemplary pro-IL18 on NK cell expansion in PBMCs. The frequency and number of NK cells were measured after PBMCs from healthy human donors were incubated with the test samples described below for 12 days. NK cells were defined as CD56+ / CD3- cells. The antibody fusion proteins tested were Fuse926 (black rhombus), composed of NKG2D xROR1 bsAb, and Fuse916 (black triangle), composed of the same bsAb but also incorporating IL-15m9-1 and pro-IL18 (granulase B-cleavable IL18mutAV). Fuse923 (black square), a cytokine bsAb fusion that targets the δ2 ​​region of the γ9δ2 TCR similarly to Fuse923, was used as a non-targeting control, and anti-HEL (gray circle) was used as an isotype-matched negative control. Notably, all bsAbs contained the LALA mutation and therefore neither bound to nor activated CD16 expressed on NK cells. Therefore, NK cell expansion is limited to targeting cytokines to NKG2D without co-linking NKG2D and CD16, which has been reported to enhance the activity of NKG2D and CD16 against each other (www.sciencedirect.com / science / article / pii / S0006497120678016). Figure 5A and Figure 5B The xy plots showing the percentage and number of NK cells measured on day 12 are presented as a function of titration concentration for each assay. 100 nM Fuse916 induced approximately 6-fold NK cell expansion compared to Fuse926.

[0466] Example 5 As shown in Figure 6, we evaluated the effect of targeting NKp46 with the exemplary IL-15 variant and the exemplary pro-IL18 on NK cell expansion in PBMCs. The frequency and number of NK cells were measured after PBMCs from healthy human donors were incubated with the test samples described below for 12 days. NK cells were defined as CD56+ / CD3- cells. The antibody fusion protein tested was Fuse943 (black triangle), which consists of a monovalent NKp46Ab incorporating IL-15m9 and pro-IL18 (a granzyme B-cleavable IL18mutAV). Fuse944 (black square) served as a non-targeting control, where the NKp46 binder was omitted from the construct. Fuse320 (black rhombus), an NKp46 x EGFR bsAb incorporating the same NKp46 binder but without cytokines, served as a targeting / cytokine-free control (note that we did not detect EGFR expression in the PBMCs; data not shown), and anti-HEL (gray circle) served as an isotype-matched negative control. Notably, all bsAbs contain the LALA mutation, thus neither binding to nor activating CD16 expressed on NK cells. Therefore, NK cell expansion is limited to targeting NKp46 with cytokines, without co-linking NKp46 and CD16, which has been reported to enhance the activity of NKG2D and CD16 against each other (www.sciencedirect.com / science / article / pii / S0006497120678016). Figure 6A and Figure 6B The xy plots showing the percentage and number of NK cells measured on day 12 are presented as a function of titration concentration for each assay. 100 nM Fuse943 induced approximately 5-fold NK cell expansion relative to Fuse320.

[0467] Example 6 As shown in Figure 7, we evaluated the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via the Vγ9Vδ2 TCRx ROR1 bsAb cytokine fusion to induce γ9δ2 T cell proliferation in human PBMCs.

[0468] After incubating PBMCs from healthy human donors with the following test samples for 12 days, the frequency and number of two γ9δ2 cell types were measured. Given that ROR1 is not expressed on any cells in the PBMCs (note that ROR1 is expressed on pre-B cells in human bone marrow), the observed expansion was considered unrelated to Vγ9Vδ2 TCR crosslinking via ROR1.

[0469] The antibody fusion protein tested was a cytokine fusion with Vγ9Vδ2 TCR x ROR1 bsAb (Fuse966; black rhombus) incorporating (1) IL-15m9-1 (Fuse968; black square), (2) pro-IL18 (granulase B-cleavable IL18mutAV) (Fuse967; black inverted triangle), and (3) both IL-15m9-1 and pro-IL18. Fuse944 (hollow triangle, dashed line) was used as a non-targeted control, in which the Vγ9Vδ2 TCR x ROR1 cassette was omitted from the construct. Anti-HEL (gray circle) was used as an isotype-matched negative control.

[0470] Figure 7A and Figure 7B The xy plots showing the percentage and number of γ9δ2 T cells measured on day 12 are presented as a function of titration concentration for each assay. 100 nM of Fuse923 induced approximately 12-fold γ9δ2 T cell expansion relative to Fuse966.

[0471] Example 7 As shown in Figure 8, we evaluated the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via a Vγ9Vδ2 TCRx ROR1 bsAb cytokine fusion to induce γ9δ2 T cell-mediated IFNγ release and tumor cell killing. The assay was designed as follows: γ9δ2 T cells enriched / expanded from healthy human donor PBMCs were mixed with ROR1+ tumor cells, stably Egfp-expressing MDA-MB-231, and firefly luciferase at a 5:1 E:T ratio. Forty-eight hours later, tumor cell cytotoxicity was assessed by luciferase activity (i.e., tumor cell viability), and IFNγ release in the supernatant was measured using a standard ELISA protocol. The antibody fusion protein tested was a cytokine fusion with Vγ9Vδ2 TCR x ROR1bsAb (Fuse966; hollow inverted triangle; dashed line), incorporating (1) IL-15m9-1 (Fuse968; hollow triangle; dashed line), (2) pro-IL18 (granulase B-cleavable IL18mutAV) (Fuse967; black inverted triangle), and (3) IL-15m9-1 and pro-IL18. Fuse944 (black diamond) was used as a non-targeted control, in which the Vγ9Vδ2 TCR x ROR1 cassette was omitted from the construct. Anti-HEL (gray circle) was used as an isotype-matched negative control. A non-linear xy plot showing the variation of tumor cell killing with titration concentration of each test sample is shown. Figure 8A), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 8B Compared to Fuse966, incorporating cytokines into bsAbs enhanced both tumor cell killing and IFNγ release, with the order being Fuse923 > Fuse967 > Fuse968 > Fuse966, indicating that the combination of IL-18 and IL-15 is most effective. The differences in IFNγ release readings between the test samples were more pronounced compared to tumor cell killing. This is likely due to the high E:T ratio of 5:1, at which maximum tumor cell killing is achieved when the γ9δ2 T cell signaling threshold is below IFNγ release. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0472] Example 8 As shown in Figure 9, we evaluated the effect of targeting NKp30 (with or without NKp46) with exemplary IL-15 and IL-18 variants on NK cells expanded from PBMCs. The frequency and number of NK cells were measured after 21 days of incubation of PBMCs from healthy human donors with 100 pM of the test sample described below. NK cells were defined as CD56+ / TCRαβ- cells. The antibody fusion protein tested was Fuse1147 (light gray), which consists of a mortar-and-groove bispecific antibody containing a monovalent arm specific to human NKp30 (Fab) and a monovalent arm specific to human ROR1 (VHH), both located at the N-terminus of the mortar (or groove). The ROR1-specific VHH was fused to the C-terminus of the NKp30-specific LC. Fuse1148 (dark gray) is identical to Fuse1147 except that it contains an NKp46-specific VHH at the C-terminus of the CH3 domain (mortar or groove). Fuse1145 (black dot) is the same as Fuse1147, except that it contains an exemplary IL-18 variant at the N-terminus of the mortar (or pestle, if the NKp30-specific fab is on the mortar) and IL-15m10-1 at the C-terminus of the mortar (or pestle). Fuse1146 (black stripe) is the same as Fuse1148, except that it contains an exemplary IL-18 variant at the N-terminus of the mortar (or pestle, if the NKp30-specific fab is on the mortar) and IL-15m10-1 at the C-terminus of the mortar (or pestle, if the NKp46-specific VHH is on the mortar). Therefore, for simplification, Fuse1147, Fuse118, Fuse1145 and Fuse1148 are respectively composed of NKp30Xror1, NKp30Xror1xNKp46, NKp30Xror1-IL-18 / IL-15 and NKp30Xror1xNKp46-IL-18 / IL-15. Figure 9A The image shows a flow cytometry dot plot of CD56 and TCRαβ. A box is drawn around NK cells (CD56+ / TCRαβ- cells), and the frequency of these cells is shown to the left of the box. The data are also presented as a bar chart. Figure 9B In this study, anti-HEL specific antibody (human IgG1 LALA) was used as a negative isotype control. Fuse1145 and Fuse1146 induced approximately 8.5-fold and 13.7-fold increases in NK cell frequency, respectively, compared to the negative control and the cytokine-free connective. We also measured the total number of NK cells in PBMCs co-cultured with each test sample (100 pM). The fold increase in NK cells relative to anti-HEL observed based on this data is shown in the figure. Figure 9CIn the control group, no significant expansion was observed when using an adaptor without cytokines. In contrast, Fuse1145 and Fuse1146 induced approximately 34-fold and 69-fold expansion, respectively. The data indicate that adding NKp46 targeting NKp30 to IL-18 / IL-15 enhances the frequency and expansion of NK cells from human PBMCs.

[0473] Example 9 As shown in Figure 10, we evaluated the agonist activity of γ9δ2 TCR-specific VHH fusion with the Fc domain of human IgG1 to induce redirected lysis of expanded γ9δ2 T cells against P815 cells stably transduced with Egfp and firefly luciferase (P815). The assay was designed as follows: γ9δ2 T cells enriched / expanded from healthy human donor PBMCs were mixed with P815 at a 3:1 E:T ratio. After 24 hours, tumor cell cytotoxicity was assessed by luciferase activity (i.e., tumor cell viability). The nonlinear xy relationship of percentage kill as a function of assay concentration is illustrated in Figure 10. Figures 10A to 10B The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0474] Example 10 As shown in Figure 11, we evaluated the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target γ9δ2 T cells via a Vγ9Vδ2TCR x ROR1 bsAb cytokine fusion to induce γ9δ2 T cell-mediated IFNγ release and tumor cell killing. The assay was designed as follows: γ9δ2 T cells enriched / expanded from healthy human donor PBMCs were mixed with ROR1+ tumor cells, Jurkat cells stably expressing Egfp, and human ROR1 at a 3:1 E:T ratio. After 72 hours, tumor cell cytotoxicity was assessed by GFP fluorescence (i.e., tumor cell viability), and IFNγ release in the supernatant was measured using a standard ELISA protocol. The antibody fusion protein tested was a cytokine fusion with Vγ9Vδ2 TCR x ROR1 bsAb (Fuse1139; black square), incorporating (1) IL-15m10-1 (Fuse1137; black triangle), (2) proIL18mutA (Fuse1138; black inverted triangle), and (3) IL-15m10-1 and proIL18mutA (Fuse1136; hollow circle, dashed line). Anti-HEL (gray circle) was used as an allotype-matched negative control. A nonlinear xy plot showing the tumor cell killing effect as a function of the titration concentration of each test sample is shown. Figure 11A), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 11B Compared to Fuse1139, incorporating cytokines into bsAbs enhanced both tumor cell killing and IFNγ release, with the order being Fuse1136 > Fuse1137 > Fuse1138, indicating that the combination of IL-18 and IL-15 is most effective. The differences in IFNγ release readings between the test samples were more pronounced compared to tumor cell killing. This is likely due to the high E:T ratio of 3:1, at which maximum tumor cell killing is achieved when the γ9δ2 T cell signaling threshold is below IFNγ release. The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0475] Example 11 As shown in Figure 12, we evaluated human NKp46 ( Figures 12A to 12B ) and NKp30 ( Figure 12C The specific VHH is fused with the Fc domain of human IgG1 to induce agonist activity in expanded γ9δ2 T cells to redirect and lyse P815 cells stably transduced with Egfp and firefly luciferase (P815). The assay is designed as follows: expanded NK cells enriched / expanded from PBMCs of healthy human donors are mixed with P815 at a 1:1 E:T ratio. Figure 12A ). Figure 12B and Figure 12C The E:T ratios were 2:1 and 3:1, respectively. After 24 hours, tumor cell cytotoxicity was assessed by luciferase activity (i.e., tumor cell viability). The nonlinear xy relationship between the percentage of cell kill and the test sample concentration is illustrated in the figure. Figures 12A to 12C middle.

[0476] The summaries of EC50, Emax, and AUC (area under the curve) values ​​are shown below each xy plot.

[0477] Example 12 As shown in Figure 13, we evaluated the ability of exemplary IL-15 variants and / or exemplary pro-IL18 to target human NK cells via the NKp46 xROR1 bsAb cytokine fusion to induce NK cell-mediated IFNγ release and tumor cell killing. The assay was designed as follows: NK cells enriched / expanded from healthy human donor PBMCs were mixed with ROR1+ tumor cells, Jurkat cells stably expressing Egfp, and human ROR1 at a 3:1 E:T ratio. After 72 hours, tumor cell cytotoxicity was assessed by GFP fluorescence (i.e., tumor cell viability), and IFNγ release in the supernatant was measured using a standard ELISA protocol. The antibody fusion protein tested was a cytokine fusion with NKp46 x ROR1 bsAb (Fuse1125; black square), incorporating (1) IL-15m10-1 (Fuse1127; black triangle), (2) proIL18mutA (Fuse1126; black inverted triangle), and (3) IL-15m10-1 and proIL18mutA (Fuse1124; hollow circle, dashed line). Anti-HEL (gray circle) was used as an allotype-matched negative control. A nonlinear xy plot showing the tumor cell killing effect as a function of the titration concentration of each test sample is shown. Figure 13A ), or a nonlinear xy plot showing the variation of IFNγ release with the titration concentration of each test sample ( Figure 13B Compared to Fuse1125, incorporating cytokines into bsAbs enhanced both tumor cell killing and IFNγ release, with the order being Fuse1124 > Fuse1127 > Fuse1126, indicating that the com...

Claims

1. A fusion protein comprising a scaffold polypeptide and at least two of (a) to (c): a. One or more variants of interleukin-15 (IL-15), b. One or more of interleukin-18 (IL-18), fragments of said IL-18, IL-18 variants, or fragments of said IL-18 variants, and c. One or more receptor-binding polypeptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more polypeptides capable of binding to tumor-associated antigens (TAAs).

2. The fusion protein of claim 1, wherein the one or more polypeptides capable of binding the TAA comprises receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH.

3. The fusion protein according to claim 1, wherein the TAA comprises EGFR, HER2, or DLL.

4. The fusion protein according to claim 1, wherein the activating receptor comprises differentiation cluster (CD)3, CD16, γ9TCR, δ2 TCR or δ1 TCR, or wherein the co-stimulatory receptor comprises differentiation cluster (CD)137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L or CD40.

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

6. The fusion protein according to any one of claims 1 to 5, wherein the fusion protein further comprises a receptor-binding polypeptide capable of binding to an activating receptor and / or a co-stimulatory receptor expressed on immune cells. Optionally, the activated receptor includes differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, and Optionally, the co-stimulatory receptors include differentiation cluster (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, 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 (ROR1) VHH are each independently fused to the C-terminus 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 (ROR1) VHH are each independently fused to the N-terminus of the scaffold polypeptide.

9. The fusion protein of claim 1, wherein the scaffold polypeptide is an antibody, 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 tyrosine kinase-like orphan receptor 1 (ROR1) VHH are each independently fused to either the C-terminus or N-terminus of the heavy chain or the light chain, or to the C-terminus of the antibody. H 2. Merge either the structural domain or the hinge region.

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 an anti-PD-L1 antibody.

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

14. The fusion protein of claim 13, wherein the crystallizable fragment (Fc) region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1.

15. The fusion protein of claim 13, wherein the crystallizable fragment (Fc) region is a mortar (KiH) Fc.

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

17. The fusion protein of claim 1, wherein the scaffold polypeptide is selected from crystallizable fragment (Fc) regions, human serum albumin (HSA), β2 microglobulin, transferrin, fragment antigen-binding regions (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalin, designed ankylosing repeat protein (DARPin), their binding domains, and fragments thereof.

18. The fusion protein according to any one of claims 1 to 17, wherein the IL-15 variant comprises the sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKEFLQSX 103 VHIVX 108 MFIX 112 TS, where a.X1 can be any amino acid. b.X4 can be any amino acid. cX 22 It can be any amino acid. dX 30 It can be any amino acid. eX 41 It can be any amino acid. fX 45 It can be any amino acid. gX 49 It is any amino acid except V. hX 53 It is any amino acid except E. iX 64 It can be any amino acid. jX 67 It can be any amino acid. kX 68 It can be any amino acid. lX 79 It can be any amino acid. mX 93 It can be any amino acid. nX 103 It can be any amino acid. oX 108 It is any amino acid, and pX 112 It is any amino acid; (SEQ ID NO:533).

19. The fusion protein according to any one of claims 1 to 18, wherein the IL-18 variant comprises: a1.MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250) has amino acid positions 37-193, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250, or a2.MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251) has amino acid positions 37-193, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251, and optionally also includes amino acid substitutions at one or more of C74, C104, C112, and C164, each independently substituted with valine, alanine, or serine, or a3. Selected from IL-18 variants in Table 6B; and b1. Optionally, the one to five amino acid substitutions are one or more of the following: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; or M149V or M149I; or b2. Optionally, 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.

20. The fusion protein according to any one of claims 1 to 19, wherein the IL-18, the fragment of the IL-18, the variant of the IL-18, or the fragment of the variant of the IL-18 further comprises its propeptide (PP) or a PP variant, and optionally, wherein the PP or the PP variant is located at the N-terminus relative to the IL-18, the fragment of the IL-18, the variant of the IL-18, or the fragment of the variant of the IL-18.

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

22. The fusion protein according to any one of claims 1 to 21, wherein 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.

23. The fusion protein of claim 22, wherein the one or more cleavage sites are located at... Between the IL-18, a fragment of the IL-18, a variant of the IL-18, or a fragment of a variant of the IL-18 and the scaffold polypeptide; or Within the PP, between the PP or a variant of the PP and the IL-18, a fragment of the IL-18, a variant of the IL-18, 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 the IL-18, a variant of the IL-18, or a fragment of the IL-18 variant, or within the PP; or Their combination.

24. The fusion protein according to any one of claims 2 to 23, wherein 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 The variants of the polypeptide having SEQ ID NO:325 (CDR1 of 2A11), the variants of the polypeptide having SEQ ID NO:326 (CDR2 of 2A11), the variants of the polypeptide having SEQ ID NO:327 (CDR3 of 2A11), or combinations thereof, wherein the variant of the polypeptide having SEQ ID NO:325 comprises one or more deletions, additions, or substitutions of amino acid residues 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 substitutions of amino acid residues in the polypeptide having SEQ ID NO:326, and wherein the variant of the polypeptide having SEQ ID NO:327 comprises one or more deletions, additions, or substitutions of amino acid residues in the polypeptide having SEQ ID NO:327, and in: The variants of the polypeptide having SEQ ID NO:325 and the variants of the polypeptide having SEQ ID NO:327 do not replace the cysteine ​​residues in the polypeptide having SEQ ID NO:325 and the polypeptide having SEQ ID NO:327, or The variants of the polypeptide having SEQ ID NO:325 and the variants of the polypeptide having SEQ ID NO:327 replace one or two cysteine ​​residues in the polypeptide having SEQ ID NO:325 and / or one or two cysteine ​​residues in the polypeptide having SEQ ID NO:327 with amino acids containing cross-linking functional groups.

25. A polynucleotide encoding a fusion protein according to any one of claims 1 to 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 the cell is a bacterial cell or a yeast cell.

31. A method for producing a fusion protein, the method comprising: The cells of any one of claims 27 to 30 are cultured in a cell culture medium to allow the production of the fusion protein, and optionally secreted into the cell culture medium.

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

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

34. A method for activating and promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, the method comprising: The fusion protein of any one of claims 1 to 24 shall be administered to a subject in need.

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

36. A method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising: The fusion protein of any one of claims 1 to 24 shall be administered to a subject in need.

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

38. A method for activating or promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, said method comprising administering to a subject in need at least two of the following: a. Interleukin-15 (IL-15) variants or IL-15 fusion proteins, b. Interleukin-18 (IL-18), a fragment of said IL-18, an IL-18 variant, a fragment of said IL-18 variant, or an IL-18 fusion protein. c. A multispecific antibody construct, wherein the multispecific antibody construct comprises: i. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH, and ii. Receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells. Optionally, the activated receptor includes differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, and Optionally, the co-stimulatory receptors include differentiation cluster (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40, and d. One or more receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more peptides capable of binding to tumor-associated antigens (TAAs).

39. The method of claim 38, wherein the subject has cancer.

40. A method for alleviating, suppressing, or treating a disease or symptom in a subject in need, said method comprising administering to the subject in need at least two of the following: a. Interleukin-15 (IL-15) variants or IL-15 fusion proteins, b. Interleukin-18 (IL-18), a fragment of said IL-18, an IL-18 variant, a fragment of said IL-18 variant, or an IL-18 fusion protein. c. A multispecific antibody construct, wherein the multispecific antibody construct comprises: i. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) VHH, and ii. Receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells. Optionally, the activated receptor includes differentiation cluster (CD)3, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, and Optionally, the co-stimulatory receptors include differentiation cluster (CD) 137, CD28, DNAM-1, NKp46, NKG2D, NKp30, CD2, ICOS, OX40, CD40L, or CD40, and d. One or more receptor-binding peptides capable of binding to activating receptors and / or co-stimulatory receptors expressed on immune cells, or one or more peptides capable of binding to tumor-associated antigens (TAAs).

41. The method of claim 40, wherein the disease or symptom is cancer.

42. A variant of interleukin-15 (IL-15), said IL-15 variant comprising the sequence of formula I: X1WVX4VISDLKKIEDLIQSMHIX 22 ATLYTESX 30 VHPSCKVTAMX 41 CFLX 45 ELQX 49 ISLX 53 SGDASIHDTVX 64 NLX 67 X 68 LANNSLSSNGX 79 VTESGCKECEELEX 93 KNIKEFLQSX 103 VHIVX 108 MFIX 112 TS, where a.X1 can be any amino acid. b.X4 can be any amino acid. cX 22 It can be any amino acid. dX 30 It can be any amino acid. eX 41 It can be any amino acid. fX 45 It can be any amino acid. gX 49 It is any amino acid except V. hX 53 It is any amino acid except E. iX 64 It can be any amino acid. jX 67 It can be any amino acid. kX 68 It can be any amino acid. lX 79 It can be any amino acid. mX 93 It can be any amino acid. nX 103 It can be any amino acid. oX 108 It is any amino acid, and pX 112 It is any amino acid; (SEQ ID NO:533).

43. The IL-15 variant of claim 42, wherein a.X1 is N (SEQ ID NO:534). b.X4 is N (SEQ ID NO:535). cX 22 It is D (SEQ ID NO:536). dX 30 It is D (SEQ ID NO:537). eX 41 It is K (SEQ ID NO:538). fX 45 It is L (SEQ ID NO:539). gX 64 It is E (SEQ ID NO:540). hX 67 It is I (SEQ ID NO:541). i.X 68 is I (SEQ ID NO: 542), jX 79 It is N (SEQ ID NO:543). kX 93 It is E (SEQ ID NO:544). lX 103 It is F (SEQ ID NO:545). mX 108 It is Q (SEQ ID NO:546), or nX 112 It is N (SEQ ID NO:547), or Any two or more items from oa to o.

44. The IL-15 variant of claim 42, wherein a.X1 is N (SEQ ID NO:534). b.X4 is N (SEQ ID NO:535). cX 22 It is D (SEQ ID NO:536). dX 30 It is D (SEQ ID NO:537). eX 41 It is K (SEQ ID NO:538). fX 45 It is L (SEQ ID NO:539). gX 49 It is R or K (SEQ ID NO:548). hX 53 It is G, K, I or A (SEQ ID NO:549). iX 64 It is E (SEQ ID NO:540). jX 67 It is I (SEQ ID NO:541). kX 68 It is I (SEQ ID NO:542). lX 79 It is N (SEQ ID NO:543). mX 93 It is E (SEQ ID NO:544). nX 103 It is F (SEQ ID NO:545). oX 108 It is Q (SEQ ID NO:546), or pX 112 It is N (SEQ ID NO:547), or Any two or more items from qa to p.

45. The IL-15 variant of claim 42, wherein a.X1 is N or G (SEQ ID NO:550). b.X4 is N, K, or L (SEQ ID NO:551). cX 22 It is either D or A (SEQ ID NO:552). dX 30 It is D or N (SEQ ID NO:553). eX 41 It is K or Q (SEQ ID NO:554). fX 45 It is L or S (SEQ ID NO:555). gX 49 It is R or K (SEQ ID NO:548). hX 53 It is G, K, I or A (SEQ ID NO:549). iX 64 It is either E or Q (SEQ ID NO:556). jX 67 It is I or T (SEQ ID NO:557). kX 68 It is I or S (SEQ ID NO:558). lX 79 It is N or Y (SEQ ID NO:559). mX 93 It is either E or A (SEQ ID NO:560). nX 103 It is F or L (SEQ ID NO:561). oX 108 Is it Q or T (SEQ ID NO:562), or pX 112 It is N or R (SEQ ID NO:563), or Any two or more items from qa to p.

46. ​​The IL-15 variant of claim 42, wherein X 49 It is R or K, and X 53 It is G, K, I or A; (SEQ ID NO:564).

47. The IL-15 variant according to claim 42, X 30 It is N (SEQ ID NO:565).

48. The IL-15 variant of claim 42, wherein X 64 It is Q (SEQ ID NO:566).

49. The IL-15 variant of claim 42, wherein X1 is G (SEQ ID NO:567).

50. The IL-15 variant of claim 42, wherein the IL-15 variant includes the IL-15 variants listed in Table 1A or Table 3A.

51. A fusion protein, said fusion protein comprising: The IL-15 variant according to any one of claims 42 to 50; and Scaffold peptides.

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 according to claim 51 or claim 52, wherein the scaffold polypeptide is an antibody or a fragment thereof.

54. The fusion protein according to 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 an anti-PD-L1 antibody.

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

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

58. The fusion protein of claim 56, wherein the Fc region is an Fc region derived from IgG4, KiH (KiH) Fc, or IgG1.

59. The fusion protein of claim 56, wherein the Fc region is a mortar (KiH) Fc.

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

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

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

68. A method for producing an IL-15 variant or fusion protein, the method comprising: The cells of any one of claims 64 to 67 are cultured in a cell culture medium to allow the production of the IL-15 variant or the fusion protein, 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 method for activating and promoting the differentiation or expansion of T cells, B cells, or natural killer (NK) cells, said method comprising: Administer the IL-15 variant of any one of claims 42 to 50, or the IL-15 fusion protein of any one of claims 51 to 61, to a subject in need.

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

72. A method for alleviating, suppressing, or treating a disease or symptom in a subject in need, the method comprising: Administer the IL-15 variant of any one of claims 42 to 50, or the IL-15 fusion protein of any one of claims 51 to 61, to a subject in need.

73. The method of claim 72, wherein the disease or symptom is cancer.

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