Cell signaling complexes and uses thereof

Abstract

Disclosed herein are cellular signaling complexes, such as chimeric cytokine complexes, and uses thereof. In some embodiments, a chimeric cytokine complex includes a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of engineered Fc antibody domains. Also disclosed are Fc cytokine complexes and uses thereof. In some embodiments, an Fc cytokine complex includes an engineered Fc antibody domain and one or more cytokines linked to the engineered Fc antibody domain.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 511,968, filed July 5, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to the electronic sequence list This application contains a sequence list, which was filed with the Patent Centre and is incorporated herein by reference in its entirety. The .xml copy was created on May 15, 2024, and is named 04_NNTNZ00100WO_sequence_listing.xml, and has a size of 35,143 bytes. Technical Field

[0003] This disclosure generally relates to the field of cell signaling complexes, and more specifically, to cell signaling complexes such as chimeric cytokine complexes and their uses. Background Technology

[0004] A common mechanism for activating cellular pathways from the cell surface is receptor oligomerization or receptor aggregation. For example, receptor oligomerization / aggregation can result from the co-stimulatory binding of receptors to ligands and agonistic antibodies [1] and from the binding of cell signaling molecules such as cytokines to certain cell receptors [2]. These receptors typically include extracellular domains on the cell surface, transmembrane domains across the cell membrane, and intracellular domains inside the cell. In these receptor aggregation patterns, the extracellular domain of the receptor binds to cell signaling molecules, which can cause conformational changes that allow it to translate across the cell membrane to promote activation / signaling; and / or can cause multiple receptors to oligomerize, thereby forming an intracellular signaling complex that drives activation / signaling.

[0005] As discussed earlier, cytokines are a well-known class of cell signaling proteins. The classic mechanism of cytokine activation is the dimerization of extracellular cytokine receptors, which is usually achieved by the binding of two JAK (Jenners kinase)-associated receptor subunits in a heterodimer (and sometimes homodimer) manner [2, 3]. JAK dimerization leads to phosphorylation of STAT (signal transduction and transcription activator) transcription factors, which drive cell activation through gene regulation [4].

[0006] Cytokines can be further classified into interleukins, interferons, chemokines, lymphokines, colony-stimulating factors (CSF), and tumor necrosis factor (TGF). An important family of cytokines is the common γ-chain (γc) cytokine family, which plays a crucial role in T cell proliferation and survival. Some members of the γc cytokine family include interleukin-2 (IL-2), IL-7, and IL-15.

[0007] The functional IL-2-binding IL-2 receptor signaling complex can be formed by the sequential binding / complexing of IL-2Rα, IL-2Rβ and γc subunits into a high-affinity heterotrimer (K). d ~10 -11 M) or a ~100-fold intermediate affinity dimer complex of IL-2Rβ and γc alone (K) d ~10 -9 The composition is M) [2, 5]. IL-2 has a low affinity for the IL-2Rα subunit (K). d ~10 -8 (M), but this binding does not induce signal transduction. Nascent T cells are thought to initially have low levels of IL-2Rα, but IL-2Rα expression is upregulated upon T cell receptor (TCR) activation. IL-2Rβ and γc are constitutively expressed on lymphohematopoietic cells, including low-density expression on nascent T cells [5, 6]. In the activated T cell scenario, IL-2 binds to the upregulated IL-2Rα subunit and the low-expressed intermediate-affinity IL-2Rβ and γc heterodimers, leading to lower subsequent activation or inefficient recruitment of all receptor subunits to assemble high-affinity signaling complexes. Therefore, strategies to enhance IL-2 receptor signaling may take the following forms: (i) increasing the affinity of IL-2 for the signal-transducing IL-2Rβ / γc heterodimer to effectively increase the stability of signal transduction; (ii) increasing the recruitment and assembly of all three IL-2 receptor subunits to form high-affinity signal transduction complexes; or (iii) increasing the number of effective intermediate-affinity and high-affinity signal transduction complexes on the cell surface.

[0008] Although there have been successful cytokine engineering studies aimed at modulating receptor signaling by focusing on regulating the binding affinity of IL-2 receptors [2, 6], these studies have primarily focused on directly altering amino acids in the cytokine sequence to disrupt or enhance its binding affinity to specific receptor subunits. Such studies typically require labor-intensive and time-consuming techniques such as directed evolution, X-ray crystallography, molecular dynamics simulations, and iterative cytokine-receptor interface engineering [2, 6], while actually utilizing only affinity methods. An example of these sequence-based engineering studies is the development of interleukin-2 (IL-2) variants known as “superkines” or super-interleukin-2 [6].

[0009] Therefore, a solution is needed that utilizes the various methods described above for inducing cytokine receptor signaling to increase IL-2 receptor signaling without requiring sequence-based alterations to the cytokines themselves. Furthermore, this solution should increase the affinity of cytokines for their signaling receptors while also increasing the total number of cytokine-receptor signaling complexes per T cell. Additionally, this solution should enable enhanced activation and expansion of human immune cells. Summary of the Invention

[0010] An improved cell signaling complex capable of promoting the activation and expansion of immune cells is disclosed. More specifically, a cell signaling complex capable of promoting the activation and expansion of immune cells (e.g., human donor peripheral blood T cells, human peripheral blood NK cells, etc.) is disclosed.

[0011] In some embodiments, the chimeric cytokine complex includes a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of engineered Fc antibody domains.

[0012] A method for activating and expanding immune cells is also disclosed. This method may include adding a chimeric cytokine complex to a population of immune cells. The chimeric cytokine complex may include a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of Fc antibody domains.

[0013] In some embodiments, the immune cell population can be activated and expanded in vitro. In various embodiments, the immune cell population can be human donor peripheral blood immune cells.

[0014] In various implementations, the immune cell population can be live T cells.

[0015] In various implementations, the immune cell population can be natural killer (NK) cells.

[0016] In some embodiments, the method may further include activating the T cell population with anti-CD3 and anti-CD28 T cell activating agents prior to the addition of the chimeric cytokine complex.

[0017] In various embodiments, the cytokine may be an interleukin. An interleukin may be at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0018] In various embodiments, the plurality of engineered Fc antibody domains may include six to twelve engineered Fc antibody domains that bind to a protein cage peptide. In some embodiments, a total of 12 to 24 cytokines may be linked to the plurality of engineered Fc antibody domains.

[0019] In various embodiments, each of one or more cytokines may be linked to one of the engineered Fc antibody domains via an engineered metalloproteinase resistance linker sequence. In some embodiments, the engineered metalloproteinase resistance linker sequence may be between 7 and 13 amino acid residues in length.

[0020] In various embodiments, the C-terminus of at least one engineered Fc antibody domain may be linked to the N-terminus of at least one cytokine.

[0021] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0022] In some embodiments, one of the cytokines may be interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0023] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0024] In some embodiments, the N-terminus of at least one engineered Fc antibody domain may be linked to the C-terminus of at least one cytokine.

[0025] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and at least one of the engineered Fc antibody domains may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0026] In some embodiments, one of the cytokines may be interleukin-7 (IL-7), and at least one of the engineered Fc antibody domains may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0027] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and at least one of the engineered Fc antibody domains may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0028] In various implementations, the engineered Fc antibody domain can be an engineered human Fc antibody domain.

[0029] In some implementations, the engineered human Fc antibody domain may be an engineered human IgG1 Fc antibody domain.

[0030] In various embodiments, one of the engineered human IgG1 Fc antibody domains may include an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0031] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0032] In some embodiments, the engineered human IgG1 Fc antibody domain may include the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0033] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0034] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0035] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0036] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0037] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0038] In some implementations, the Fc antibody domain may be an engineered rabbit Fc antibody domain.

[0039] In various embodiments, the chimeric cytokine complex may further include a signal peptide linked to the N-terminus of at least one engineered Fc antibody domain or at least one cytokine.

[0040] In some embodiments, the signal peptide may be a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells. In some embodiments, the signal peptide may include the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0041] In various embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in any of the sequences in SEQ ID NO:8-15.

[0042] In some embodiments, the amino acid sequence of the polypeptide constituting the protein cage polypeptide may include at least the following point mutation relative to the amino acid sequence shown in any of SEQ IDNO: 8-15: Y294A.

[0043] In some embodiments, the protein cage polypeptide may include a polypeptide comprising a binding site for engineering one of the Fc antibody domains. This binding site may include the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16).

[0044] In some embodiments, the protein cage polypeptide may include a polypeptide comprising a binding site for engineering one of the Fc antibody domains. The binding site may include the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17).

[0045] In some embodiments, the protein cage polypeptide may include a polypeptide comprising a binding site for engineering one of the Fc antibody domains. The binding site may include the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18).

[0046] In some embodiments, the protein cage polypeptide may include a polypeptide comprising a binding site for engineering one of the Fc antibody domains. The binding site may include the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19).

[0047] In some implementations, protein cage peptides can self-assemble into tetrahedral pyramidal structures.

[0048] In some embodiments, an Fc cytokine complex is disclosed, comprising an engineered Fc antibody domain and one or more cytokines linked to the engineered Fc antibody domain.

[0049] A method for activating and expanding immune cells using an Fc cytokine complex is also disclosed. This method may include adding the Fc cytokine complex to a population of immune cells. The Fc cytokine complex may include an Fc antibody domain and one or more cytokines linked to the Fc antibody domain.

[0050] In some embodiments, the immune cell population can be activated and expanded in vitro. In various embodiments, the immune cell population can be human donor peripheral blood immune cells. In various embodiments, the immune cell population can be live T cells.

[0051] In some implementations, the immune cell population may be natural killer (NK) cells.

[0052] In some embodiments, the method may further include activating the T cell population with anti-CD3 and anti-CD28 T cell activating agents prior to the addition of the Fc cytokine complex.

[0053] In various implementations, the cytokine may be interleukin.

[0054] In some embodiments, interleukin can be at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0055] In various embodiments, each of one or more cytokines may be linked to an engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence. In some embodiments, the engineered metalloproteinase resistance linker sequence may be between 7 and 13 amino acid residues in length.

[0056] In various implementations, the C-terminus of the engineered Fc antibody domain can be linked to the N-terminus of at least one cytokine.

[0057] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and the engineered Fc antibody domain may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0058] In some embodiments, one of the cytokines may be interleukin-7 (IL-7), and the engineered Fc antibody domain may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0059] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and the engineered Fc antibody domain may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0060] In various implementations, the N-terminus of the engineered Fc antibody domain can be linked to the C-terminus of at least one cytokine.

[0061] In some embodiments, one of the cytokines may be interleukin-2 (IL-2), and the engineered Fc antibody domain may be linked to IL-2 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0062] In some embodiments, one of the cytokines may be interleukin-7 (IL-7), and the engineered Fc antibody domain may be linked to IL-7 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0063] In some embodiments, one of the cytokines may be interleukin-15 (IL-15), and the engineered Fc antibody domain may be linked to IL-15 via an engineered metalloproteinase resistance linker sequence including the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0064] In various embodiments, the engineered Fc antibody domain may be an engineered human Fc antibody domain. In some embodiments, the engineered human Fc antibody domain may be an engineered human IgG1 Fc antibody domain.

[0065] In some embodiments, the engineered human IgG1 Fc antibody domain may include an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0066] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0067] In some embodiments, the engineered human IgG1 Fc antibody domain may include the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0068] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0069] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0070] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0071] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0072] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0073] In some implementations, the Fc antibody domain may be an engineered rabbit Fc antibody domain.

[0074] In various embodiments, the Fc cytokine complex may further include a signal peptide linked to the N-terminus of an engineered Fc antibody domain or at least one cytokine. In some embodiments, the signal peptide may be a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells. In some embodiments, the signal peptide may include the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26). Attached Figure Description

[0075] Figure 1A and Figure 1B The predicted structure is illustrated, representing one implementation of an engineered Fc antibody domain linked to multiple cytokines.

[0076] Figure 1C The predicted structure of one embodiment of an engineered self-assembled protein cage peptide is illustrated.

[0077] Figure 1D The predicted structure of one embodiment of a cytokine (e.g., IL-2) and its receptor signaling complex is illustrated.

[0078] Figure 1EA predicted structure of one embodiment of a chimeric cytokine complex (CCC) is illustrated, the chimeric cytokine complex comprising an Fc cytokine bound to an engineered self-assembled protein cage polypeptide and a receptor signaling complex bound to the cytokine of the CCC.

[0079] Figure 2 This illustrates how the C-terminus or N-terminus of engineered Fc antibody domains can be linked to cytokines to enhance their activation and expansion of T cells.

[0080] Figure 3 Examples of adapter sequences designed to connect Fc and cytokine components of various CCCs are shown.

[0081] Figure 4 An example of an engineered Fc component of a CCC stabilized by a neighboring cell's Fcγ receptor (FcγR) is illustrated.

[0082] Figure 5 This illustrates how the Fc component of CCC can be engineered by mutating key sites in its FcγR binding region.

[0083] Figure 6 A graph illustrating the results of an ELISA specifically designed to detect IFN-γ produced by T cells is shown below. Phosphate-buffered saline (PBS) solution was used as a control.

[0084] Figure 7 A graph illustrating the live T cell population as determined by flow cytometry on day 7 is shown. T cells were activated and expanded using CCC, Fc-IL-2, and IL-2 alone, respectively. Phosphate-buffered saline (PBS) solution was used as a control.

[0085] Figure 8 A graph illustrating the viable T cell population as measured by flow cytometry on day 7 is shown. T cells were activated and expanded using a combination of CCC and cytokines, as well as cytokines alone. Phosphate-buffered saline (PBS) was used as a control.

[0086] Figures 9A-9C The graphs illustrate the CD4+ T cell count, CD8+ T cell count, and total viable T cells on day 7, exemplified by T cells activated and expanded by CCC and cytokines alone. Phosphate-buffered saline (PBS) solution was used as a control.

[0087] Figure 10 To illustrate the following graph: stimulation with anti-CD3 and anti-CD28 T cell activating agents upregulated the expression of cytokines and chemokines in peripheral blood donor T cells, which increased the expression of certain matrix metalloproteinases (MMPs) in T cells.

[0088] Figures 11A-11D As an example, NK cells on a medium supplemented with CCC proliferated significantly more after three days of co-culture than NK cells on a medium supplemented with either standard soluble cytokines or Fc cytokines alone. Detailed Implementation

[0089] Figure 1A and Figure 1B The predicted structure is illustrated, representing one implementation of an engineered Fc antibody domain linked to multiple cytokines. For example, Figure 1A An example is shown of a predicted structure of an engineered Fc antibody domain (e.g., an engineered IgG1 Fc heavy chain) with its C-terminus linked to two IL-2 cytokines. Another example is... Figure 1B The predicted structure of an engineered Fc antibody domain (e.g., an engineered IgG1 Fc heavy chain) is illustrated, with the N-terminus of the domain linked to two IL-2 cytokines.

[0090] In some implementations, the engineered Fc antibody domain may be an engineered human Fc antibody domain. For example, the Fc antibody domain may be an engineered human IgG1 Fc antibody domain.

[0091] In some embodiments, the engineered human IgG1 Fc antibody domain may include an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1 (see Table 1).

[0092] In other embodiments, the Fc antibody domain may be an engineered rabbit Fc antibody domain. For example, the Fc antibody domain may be an engineered rabbit IgG Fc antibody domain.

[0093] although Figure 1A and Figure 1B The cytokine IL-2 is exemplified, but this disclosure contemplates other interleukins, such as IL-7 and IL-15 (see, for example, Figure 3 and Figure 6 It can also act as a cytokine. Furthermore, this disclosure envisions other receptor-binding molecules (engineered or natural) that could be used as cellular signaling components of CCC.

[0094] Figure 1C The predicted structures of engineered self-assembled protein cage peptides are illustrated. For example... Figure 1C As shown, this protein cage polypeptide can self-assemble into a tetrahedral pyramidal structure. It can also self-assemble into a compact asymmetric multimer structure or cage-cage multimers (including dimers).

[0095] The protein cage polypeptide can be any of the protein cage polypeptides or scaffold proteins discussed in U.S. Patent Publication No. 2022 / 0196655 (the contents of which are incorporated herein by reference in their entirety).

[0096] This engineered self-assembling protein cage peptide can serve as a carrier or scaffold for multiple engineered Fc antibody domains, each domain linked to one or more cytokines. When multiple engineered Fc antibody domains (each linked to one or more cytokines) bind to this protein cage peptide, this structure is referred to herein as a chimeric cytokine complex (CCC).

[0097] Figure 1D Examples of cytokines (e.g., IL-2) and their receptor signaling complexes are illustrated. In some embodiments, the receptor signaling complex may include IL-2Rα, IL-2Rβ, and γc. In other embodiments, the receptor complex may be a dimer complex of only IL-2Rβ and γc.

[0098] Figure 1E An example of a predicted structure for a CCC is illustrated, which includes an Fc cytokine binding to an engineered self-assembled protein cage polypeptide and a receptor signaling complex binding to the CCC's cytokine (e.g., IL-2). The protein cage polypeptide may include multiple potential binding sites for engineered Fc antibody domains. For example, the protein cage polypeptide may include up to 12 binding sites for Fc antibody domains.

[0099] although Figure 1E Only one Fc cytokine chimera (e.g., Fc-IL-2) binding to a protein cage peptide is illustrated, but this disclosure envisions that between six and twelve engineered Fc antibody domains can bind to a protein cage peptide.

[0100] Since each engineered Fc antibody domain can include up to two cytokines, each CCC can include between 12 and 24 cytokines.

[0101] Table 1 : Engineered Fc sequences and engineered Fc cytokine sequences designed and tested to date.

[0102]

[0103] The protein cage polypeptide of CCC may include a polypeptide with a length of about 400 to about 700 amino acid residues. In some embodiments, the protein cage polypeptide may include a polypeptide with a length of about 450 to about 650 amino acid residues.

[0104] In some embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the sequences shown in SEQ ID NO: 8-11 (see Table 2).

[0105] The protein cage polypeptide shown in SEQ ID NO: 12 can be designed by the applicant as a Y294A mutant of the protein cage polypeptide shown in SEQ ID NO: 8 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 13 can be designed by the applicant as a Y294A mutant of the protein cage polypeptide shown in SEQ ID NO: 9 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 14 can be designed by the applicant as a Y294A mutant of the protein cage polypeptide shown in SEQ ID NO: 10 (see Table 2). The protein cage polypeptide shown in SEQ ID NO: 15 can be designed by the applicant as a Y294A mutant of the protein cage polypeptide shown in SEQ ID NO: 11 (see Table 2).

[0106] The various protein cage peptides disclosed herein may also include peptide sequences capable of binding to engineered Fc antibody domains. For example, the protein cage peptides shown in SEQ ID NO: 8 and 12 may include the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16) (referred to herein as peptide sequence A, see Table 2) for binding to engineered Fc antibody domains.

[0107] Additionally, the protein cage peptides shown in SEQ ID NO: 9 and 13 may include the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17) (referred to herein as peptide sequence B, see Table 2) for binding to the engineered Fc antibody domain.

[0108] In addition, the protein cage peptides shown in SEQ ID NO: 10 and 14 may include the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18) (referred to herein as peptide sequence C, see Table 2) for binding to the engineered Fc antibody domain.

[0109] In addition, the protein cage peptides shown in SEQ ID NO: 11 and 15 may include the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19) (referred to herein as peptide sequence D, see Table 2) for binding to the engineered Fc antibody domain.

[0110] Table 2 : Sequences of protein cage peptide variants designed and experimentally tested to date, as well as peptide sequences capable of binding to engineered Fc antibody domains.

[0111]

[0112] Figure 2 As illustrated, the C-terminus or N-terminus of the engineered Fc antibody domain can be linked to a cytokine (which can then bind to a protein cage peptide) to achieve its enhanced function of activating and expanding T cells, surpassing the capabilities of soluble cytokines themselves. While the applicant anticipated that C-terminal-linked Fc cytokines would promote less spatially confined FcγR binding, an unexpected finding was that N-terminal-linked Fc cytokine CCC could also effectively activate and expand T cells, exceeding the capabilities of soluble cytokines.

[0113] like Figure 2 As shown, the C-terminus of the engineered Fc antibody domain of CCC can be linked to the N-terminus of at least one cytokine. Table 1 lists representative sequences of several Fc cytokine variants.

[0114] For example, when one of the cytokines is interleukin-2 (IL-2), the C-terminus of at least one engineered Fc antibody domain may be linked to the N-terminus of IL-2 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-2). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20) (see also...) Figure 3 ).

[0115] For example, when one of the cytokines is interleukin-7 (IL-7), the C-terminus of at least one engineered Fc antibody domain may be linked to the N-terminus of IL-7 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-7). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21) (see also...) Figure 3 ).

[0116] As an additional example, when one of the cytokines is interleukin-15 (IL-15), the C-terminus of at least one engineered Fc antibody domain may be linked to the N-terminus of IL-15 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-15). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence SLSPGKN (SEQ ID NO: 22) (see also...) Figure 3 ).

[0117] Figure 2 It is also illustrated that the N-terminus of the engineered Fc antibody domain can be linked to the C-terminus of at least one cytokine (which can subsequently bind to a protein cage peptide) to achieve its enhanced function of activating and amplifying T cells, surpassing the capabilities of the soluble cytokine itself. Table 1 also lists representative sequences of these Fc cytokine variants.

[0118] For example, when one of the cytokines is interleukin-2 (IL-2), the N-terminus of at least one engineered Fc antibody domain may be linked to the C-terminus of IL-2 via an engineered metalloproteinase resistance linker sequence (referred to herein as IL-2-Fc). In some embodiments, the engineered metalloproteinase resistance linker may include the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23) (see also...). Figure 3 ).

[0119] For example, when one of the cytokines is interleukin-7 (IL-7), the N-terminus of at least one engineered Fc antibody domain may be linked to the C-terminus of IL-7 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-7). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24) (see also...). Figure 3 ).

[0120] As an additional example, when one of the cytokines is interleukin-15 (IL-15), the N-terminus of at least one engineered Fc antibody domain may be linked to the C-terminus of IL-15 via an engineered metalloproteinase resistance linker sequence (referred to herein as Fc-IL-15). In some embodiments, the engineered metalloproteinase resistance linker sequence may include the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25) (see also...) Figure 3 ).

[0121] Figure 2It is also illustrated that CCC may include a signal peptide or leader sequence linked to the N-terminus of at least one engineered Fc antibody domain or at least one cytokine.

[0122] In some embodiments, the signal peptide may be a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells. This signal peptide can enhance the secretion efficiency of the engineered molecule in CHO cells.

[0123] As a more specific example, a signal peptide may include the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0124] Figure 3 Examples of adapter sequences designed to connect the Fc and cytokine components of the following CCCs are shown: (1) Fc-IL-2CCC, (2) IL-2-Fc CCC, (3) Fc-IL-7 CCC, (4) IL-7-Fc CCC, (5) Fc-IL-15 CCC, and (6) IL-15-Fc CCC (see also Table 1). Figure 3 In the text, the connector is marked with an underline.

[0125] Figure 3 Various cleavage sites are also shown (e.g., cleavage sites for aspartic proteases, cysteine ​​proteases, metalloproteinases, serine proteases, and various protease superfamilies). As will be discussed in more detail in Example 3, metalloproteinases are known for mediating the shedding of cell surface receptors or cleaving the functional domains of receptors and ligands from the cell. Figure 3 As shown, except for serine proteases, all linkers were not cleaved in the middle of the linker. Since serine proteases are typically located inside the cell itself, their impact is relatively small.

[0126] Figure 3 The amino acid sequences shown (and listed below) are portions of the amino acid sequences listed in Table 1 above: The engineered Fc-IL-2 components: QQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML (SEQ ID NO: 27).

[0127] Part of the engineered IL-2-Fc: WITFCQSIISTLTPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP (SEQ ID NO:28).

[0128] The engineered Fc-IL-7 components: SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNN (SEQ ID NO: 29).

[0129] Parts of the engineered IL-7-Fc: DLCFLKRLLQEIKTCWNKILMGTKEHPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV (SEQ ID NO: 30).

[0130] The engineered Fc-IL-15 components: WQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV (SEQ ID NO: 31).

[0131] Parts of the engineered IL-15-Fc: FVHIVQMFINTSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP (SEQ ID NO: 32).

[0132] Figure 4 An example of an engineered Fc component of a CCC stabilized by a neighboring cell's Fcγ receptor (FcγR) is illustrated. The Fc component of the CCC can be stabilized via trans-stabilization by neighboring cells expressing FcγR.

[0133] In some implementations, the Fc component of CCC can be engineered to include one or more point mutations to modulate FcγR binding. For example, the Fc component can be engineered to have FcγR binding ability (i.e., increased affinity for FcγR binding) or to lack FcγR binding ability (i.e., decreased affinity for FcγR binding) to avoid antibody-dependent cytotoxicity (ADCC) activation of NK cells.

[0134] There are three main classes of Fcγ receptors (FcγRs) (FcγRI, FcγRII, and FcγRIII) on leukocytes, which play roles in cellular processes such as ADCC, cytokine release, phagocytosis, and endocytosis [7]. The main signal that enables NK cells to initiate cytotoxic activities (such as the release of perforin and granzymes) is ADCC, which requires the aggregation of FcγRIIIa receptors on the surface of NK cells [8]. Depending on the application, NK cell-mediated ADCC may or may not be effective.

[0135] Figure 5 An example is shown where the Fc component of CCC (e.g., human IgG1 Fc) can be engineered by mutating key sites of its FcγR binding moiety. Figure 5 Various methods for modifying the FcγR binding site via point mutations are shown. Some such point mutations are also discussed in references [8, 9, and 10].

[0136] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to reduce its affinity for certain Fcγ receptors (e.g., FcγRIII) and functionally reduce ADCC: P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0137] For example, the Y80K variant can significantly reduce binding to the FcγRIIIa receptor, thereby weakening ADCC. Similarly, the Y80P mutant can significantly reduce binding to FcγRIIIa and lead to insufficient N-glycosylation.

[0138] As another example, the Y80R variant can significantly reduce binding to the FcγRIIIa receptor. Similarly, the Y80G variant can significantly reduce binding to the FcγRIIIa receptor. As yet another example, the Y80A variant can significantly reduce binding to the FcγRIIIa receptor and attenuate ADCC.

[0139] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to increase affinity for certain Fcγ receptors and functionally enhance ADCC:Y80W.

[0140] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to reduce affinity for certain Fcγ receptors (e.g., FcγRIIIa) and have a neutral effect on other Fcγ receptors (e.g., FcγRII): S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0141] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to increase affinity for certain Fcγ receptors (e.g., FcγRIIIa) and have a neutral effect on other Fcγ receptors (e.g., FcγRII): E117, K118A, and A123T.

[0142] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to increase affinity for certain Fcγ receptors (e.g., FcγRII) and decrease affinity for other Fcγ receptors (e.g., FcγRIIIa): H52A, R85A, and K106A.

[0143] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to reduce affinity for certain Fcγ receptors (e.g., FcγRII and FcγRIIIa): D54A, Q79A, and A111S.

[0144] In some embodiments, the engineered human IgG1 Fc antibody domain may include at least one of the following point mutations relative to SEQ ID NO: 1 (see Table 1) to increase affinity for certain Fcγ receptors (e.g., FcγRII and FcγRIIIa): T40A and K74A.

[0145] As discussed earlier, the Fc component of CCC can be engineered to have FcγR binding ability (i.e., increased affinity for FcγR binding) to functionally increase ADCC; or it can be made to lose FcγR binding ability (i.e., decreased affinity for FcγR binding) to functionally weaken ADCC.

[0146] Example The following examples are intended to illustrate the practice of various embodiments of this disclosure. They are not intended to limit or restrict the entire scope of this disclosure. It should be understood that this disclosure is not limited to the specific embodiments described and illustrated herein, but includes all modifications and variations falling within the scope of this disclosure as defined by the appended embodiments.

[0147] Example 1: Chimeric cytokine complex (CCC) enhances T cell activation and expansion beyond soluble cytokines. An unexpected result of the experiments disclosed in this paper is that CCC enhanced or significantly increased T cell activation and expansion compared to equal concentrations of individual soluble cytokines or even individual Fc cytokines.

[0148] CD3 in human peripheral blood + T cells were thawed and the medium was replaced in serum-free T cell expansion medium. T cells were then inoculated at a concentration of 1 × 10⁶ cells / mL. 6 Cells were seeded at a concentration of [number] cells / mL and incubated at 37°C with 5% CO2 on day -1. On day 0, cells were activated with anti-CD3 and anti-CD28 T cell activating agents. In some embodiments, the T cell activating agent may be a soluble T cell activating factor for in vitro use.

[0149] In some embodiments, the anti-CD3 and anti-CD28 T cell activating agents may include various self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. These self-assembled protein nanoparticles may include protein cage peptides assembled into a three-dimensional structure.

[0150] In some embodiments, the three-dimensional structure of the protein cage polypeptide can be a tetrahedral pyramid. The protein cage polypeptide can also self-assemble into a compact asymmetric multimer structure or cage-cage multimers (including dimers).

[0151] This three-dimensional structure (e.g., a protein cage polypeptide forming a tetrahedral pyramid) can function as a scaffold for anti-CD3 and anti-CD28 antibodies.

[0152] In some embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the sequences shown in SEQ ID NO: 8-10 (see Table 2).

[0153] Protein cage peptides may include peptides with a length of about 400 to about 700 amino acid residues. In some embodiments, protein cage peptides may include peptides with a length of about 450 to about 650 amino acid residues.

[0154] In some implementations, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD3 antibodies may be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio; (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems; (iii) MACS distributed by Miltenyi Biotec. ® GMP-purified CD3 antibody; or (iv) GMP-purified anti-human CD3 SF antibody distributed by BioLegend.

[0155] In some implementations, the anti-CD28 antibody may be an agonist clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD28 antibodies may be used: (i) anti-CD28 [YTH 913.12] antibody distributed by Absolute Antibody; (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec; (iii) anti-human CD28 antibody, clone cd28.2, purified by Ultra-LEAF™, distributed by BioLegend; or (iv) mouse anti-human CD28 antibody, clone L293, purified by BD™, distributed by BD Biosciences.

[0156] Standard soluble cytokines were added to the culture medium at concentrations of 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, and 35 ng / mL, and CCC was added to the culture medium at a concentration equivalent to the standard cytokines (molar equivalents, me). 10 ng / mL of Fc-IL-2 was also added. The added CCC was FC-IL-2 CCC. The added cytokine was soluble IL-2. The cells were then returned to a 37°C incubator with 5% CO2.

[0157] On day 5, the culture medium was collected for an ELISA that is specific for detecting interferon-γ (IFN-γ). Figure 6 The following is a graph illustrating the results of an ELISA test that is specific for detecting IFN-γ. Figure 6 As shown, T cells on culture media supplemented with CCC (e.g., FC-IL-2CCC) are significantly more activated than T cells on culture media supplemented with a single cytokine (e.g., IL-2) or even Fc cytokines (e.g., Fc-IL-2), as indicated by the IFN-γ levels produced by T cells.

[0158] Fresh cell culture medium supplemented with CCC or standard cytokines was added to the cells at equal concentrations (e.g., 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, and 35 ng / mL). The cells were then returned to a 37°C, 5% CO2 incubator. On day 7, the cells were resuspended and collected for flow cytometry.

[0159] Figure 7 A graph illustrating the viable T cell population as determined by flow cytometry on day 7 is shown as an example. Cells were stained with Zombie Aqua to determine the viable T cell population. Figure 7 As shown, on day 7, the total number of live T cells on the medium supplemented with CCC (e.g., Fc-IL-2 CCC) significantly exceeded the total number of live T cells on the medium supplemented with a single cytokine (e.g., IL-2) or even Fc cytokines (e.g., Fc-IL-2).

[0160] An unexpected result of the experiments disclosed in this paper is that CCC at concentrations between 10 ng / mL and 50 ng / mL significantly enhanced T cell proliferation compared to single soluble cytokines (e.g., IL-2) at equal or even higher concentrations.

[0161] The experiments disclosed in this paper yielded another unexpected result: the type of Fc cytokine complex disclosed in this paper (e.g., Fc-IL-2) also enhanced T cell proliferation compared to single soluble cytokines (e.g., IL-2) at equal or even higher concentrations.

[0162] Figure 8 A graph illustrating the viable T cell population as determined by flow cytometry on day 7 is shown as an example. Cells were stained with Zombie Aqua to determine the viable T cell population. Figure 8 As shown, on day 7, the total number of live T cells on the culture medium supplemented with a combination of IL-7-Fc CCC (i.e., where the N-terminus of the engineered Fc antibody domain is linked to the C-terminus of the IL-7 cytokine) and a soluble cytokine (e.g., IL-15) significantly exceeded the total number of live T cells on the culture medium supplemented with a single soluble cytokine (e.g., IL-7 and IL-15).

[0163] Example 2: Chimeric cytokine complex (CCC) increases CD4 in expanded T cells + The ability of T cell counts to exceed soluble cytokines The experiments disclosed in this paper yielded another unexpected result: CCC significantly increased CD4 in expanded T cells. + content. Figures 9A-9C The CD4 values ​​for day 7 are shown below.+ T cell count, CD8 + T cell count and total live T cells.

[0164] Human peripheral blood T cells were thawed and the culture medium was replaced in serum-free T cell expansion medium. The T cells were then inoculated at a concentration of 1 × 10⁶ cells / mL. 6 Cells were seeded at a concentration of [number] cells / mL and incubated at 37°C with 5% CO2 on day -1. On day 0, cells were activated with anti-CD3 and anti-CD28 T cell activating agents.

[0165] In some embodiments, the anti-CD3 and anti-CD28 T cell activating agents may include various self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. These self-assembled protein nanoparticles may include protein cage peptides assembled into a three-dimensional structure.

[0166] In some embodiments, the three-dimensional structure of the protein cage polypeptide can be a tetrahedral pyramid. The protein cage polypeptide can also self-assemble into a compact asymmetric multimer structure or cage-cage multimers (including dimers).

[0167] This three-dimensional structure (e.g., a protein cage polypeptide forming a tetrahedral pyramid) can function as a scaffold for anti-CD3 and anti-CD28 antibodies.

[0168] In some embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the sequences shown in SEQ ID NO: 8-10 (see Table 2).

[0169] Protein cage peptides may include peptides with a length of about 400 to about 700 amino acid residues. In some embodiments, protein cage peptides may include peptides with a length of about 450 to about 650 amino acid residues.

[0170] In some implementations, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD3 antibodies may be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio; (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems; (iii) MACS distributed by Miltenyi Biotec. ® GMP-purified CD3 antibody; or (iv) GMP-purified anti-human CD3 SF antibody distributed by BioLegend.

[0171] In some implementations, the anti-CD28 antibody may be an agonist clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD28 antibodies may be used: (i) anti-CD28 [YTH 913.12] antibody distributed by Absolute Antibody; (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec; (iii) anti-human CD28 antibody, clone cd28.2, purified by Ultra-LEAF™, distributed by BioLegend; or (iv) mouse anti-human CD28 antibody, clone L293, purified by BD™, distributed by BD Biosciences.

[0172] Standard soluble cytokines were added to the culture medium at a concentration of 20 ng / mL, and CCCs were added to the medium at a concentration equivalent to the standard cytokines (molar equivalents, me). The added CCC was IL-2-Fc CCC. Soluble IL-2 was added for comparison. On day 7, cells were resuspended and collected for flow cytometry. Cells were stained with Zombie Aqua to determine the viable cell population. T cells were then run on a flow cytometer.

[0173] Example 3: Multiplex assay of cytokines and chemokines revealed increased expression of matrix metalloproteinases (MMPs) in T cells. CD3 in human peripheral blood + T cells were thawed and the medium was replaced in serum-free T cell expansion medium. T cells were then inoculated at a concentration of 1 × 10⁶ cells / mL. 6 GREX cells were seeded at a concentration of 1 cell / mL. ® In 24-well plates, the cells were incubated at 37°C with 5% CO2 on day -1. On day 0, T cells were activated with anti-CD3 and anti-CD28 T cell activation reagents.

[0174] In some embodiments, the anti-CD3 and anti-CD28 T cell activating agents may include various self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. These self-assembled protein nanoparticles may include protein cage peptides assembled into a three-dimensional structure.

[0175] In some embodiments, the three-dimensional structure of the protein cage polypeptide can be a tetrahedral pyramid. The protein cage polypeptide can also self-assemble into a compact asymmetric multimer structure or cage-cage multimers (including dimers).

[0176] This three-dimensional structure (e.g., a protein cage polypeptide forming a tetrahedral pyramid) can function as a scaffold for anti-CD3 and anti-CD28 antibodies.

[0177] In some embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the sequences shown in SEQ ID NO: 8-10 (see Table 2).

[0178] Protein cage peptides may include peptides with a length of about 400 to about 700 amino acid residues. In some embodiments, protein cage peptides may include peptides with a length of about 450 to about 650 amino acid residues.

[0179] In some implementations, the anti-CD3 antibody may be an "OKT3" clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD3 antibodies may be used: (i) anti-CD3 monoclonal antibody (OKT3) distributed by Takara Bio; (ii) GMP monoclonal anti-human CD3 antibody (OKT3) distributed by ACROBiosystems; (iii) MACS distributed by Miltenyi Biotec. ® GMP-purified CD3 antibody; or (iv) GMP-purified anti-human CD3 SF antibody distributed by BioLegend.

[0180] In some implementations, the anti-CD28 antibody may be an agonist clone having multiple host isotypes (such as human, mouse, rabbit, etc.). For example, any of the following anti-CD28 antibodies may be used: (i) anti-CD28 [YTH 913.12] antibody distributed by Absolute Antibody; (ii) anti-human CD28 antibody, clone 15E8, distributed by Miltenyi Biotec; (iii) anti-human CD28 antibody, clone cd28.2, purified by Ultra-LEAF™, distributed by BioLegend; or (iv) mouse anti-human CD28 antibody, clone L293, purified by BD™, distributed by BD Biosciences.

[0181] Cytokines (e.g., IL-2) were added to the culture medium to achieve a final concentration of 10 ng / mL. 500 µL of culture medium without IL-2 was frozen and used as a control sample on day 0. The T cells were then returned to an incubator at 37°C with 5% CO2. On day 3, 500 µL of culture medium was collected from each well and frozen. The used culture medium was then replaced with fresh culture medium supplemented with IL-2 to achieve a final concentration of 10 ng / mL. The T cells were then returned to an incubator at 37°C with 5% CO2. On day 7, 500 µL of culture medium was collected from each well and frozen. The used culture medium was then replaced with fresh culture medium supplemented with IL-2 to achieve a final concentration of 10 ng / mL. The T cells were then returned to an incubator at 37°C with 5% CO2. Samples frozen on days 0, 3, and 7 were subjected to a multiplex detection panel for 71 human cytokines / chemokines. The panel used a combination of antibody-conjugated fluorescent beads to capture the analytes and used fluorescent detection antibodies to determine the concentration of cytokines and chemokines in the culture medium.

[0182] like Figure 10 As shown in the chart, stimulation with anti-CD3 and anti-CD28 T cell activating agents upregulated the expression of cytokines and chemokines in peripheral blood donor T cells, which increased the expression of matrix metalloproteinases (MMPs) (also known as matrix metalloproteinases) in T cells.

[0183] This is consistent with other studies that have shown T cells and NK cells express MMPs and that the receptor CD100 can be “shed” from the cell surface via MMP proteolytic activity [11, 12]. Furthermore, chemokines and cytokines, TNF-α, IL-1, MIP-1α, MIP-1β, and RANTES, have been shown to upregulate CD3+. + and CD4 + T cells secrete proMMP-9 (a protease precursor that is cleaved into active MMP-9) [13, 14]. In addition, IL-2 stimulation increases the production of MMP-9 in T cells

[11] .

[0184] Linking these research directions with studies on cell activating effects based on CCC and Fc cytokines necessitates protecting the chimeric Fc cytokine linker of the CCC and Fc cytokine complex from metalloproteinases.

[0185] Therefore, this article discloses that the CCC and Fc cytokine complex possesses a chimeric Fc cytokine linker or a linker sequence that protects it from metalloproteinases (see [link]). Figure 2 and Figure 3). Such linkers are designed to be free of any predicted metalloproteinase sites. Part of this work involves the use of a protease site prediction tool called PROSPER

[15] .

[0186] One of the technical challenges faced by the applicant is how to engineer metalloproteinase resistance linkers that promote agonistic or receptor binding. One technical solution discovered and developed by the applicant is to engineer metalloproteinase resistance linkers such that the linker sequence includes amino acid residues of 7 to 13 in length. Literature supports this approach, showing that flexibility is inversely proportional to agonistic activity in the case of FcγR

[16] . The applicant predicts that a similar relationship will exist for Fc cytokine chimeras, since both antibodies [1] and cytokine agonism [2,3] involve similar types of receptor aggregation mechanisms.

[0187] In some embodiments, the anti-CD3 and anti-CD28 T cell activating agents may include various self-assembled protein nanoparticles modified with anti-CD3 and anti-CD28 antibodies. These self-assembled protein nanoparticles may include protein cage peptides assembled into a three-dimensional structure.

[0188] In some embodiments, the three-dimensional structure of the protein cage polypeptide can be a tetrahedral pyramid. The protein cage polypeptide can also self-assemble into a compact asymmetric multimer structure or cage-cage multimers (including dimers).

[0189] This three-dimensional structure (e.g., a protein cage polypeptide forming a tetrahedral pyramid) can function as a scaffold for anti-CD3 and anti-CD28 antibodies.

[0190] In some embodiments, the protein cage polypeptide may include a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the sequences shown in SEQ ID NO: 8-10 (see Table 2).

[0191] Protein cage peptides may include peptides with a length of about 400 to about 700 amino acid residues. In some embodiments, protein cage peptides may include peptides with a length of about 450 to about 650 amino acid residues.

[0192] Example 4: Chimeric cytokine complex (CCC) enhances NK cell activation and expansion Another unexpected result from the experiments disclosed in this paper is that CCC enhanced or significantly increased NK cell activation and expansion compared to equal concentrations of individual soluble cytokines or individual Fc cytokines.

[0193] On day 0, human peripheral blood CD56+ NK cells were thawed and the medium was replaced in serum-free NK cell expansion medium. This medium was supplemented with 10% human platelet lysate and one of the following: (1) a standard soluble cytokine (e.g., IL-2), (2) an Fc cytokine complex (e.g., IL-2-Fc), (3) a CCC (e.g., IL-2-Fc), and (4) a CCC containing an NK activating factor (e.g., IL-2-Fc). For example, the NK activating factor could be a soluble NK activating factor for in vitro use.

[0194] NK cells were 7 × 10 5 Cells were seeded at a concentration of [number] cells / mL and then incubated at 37°C with 5% CO2. On day 3, the cells were imaged under an optical microscope.

[0195] Standard soluble cytokines were added to the culture medium at a concentration of 35 ng / mL, and CCC was added to the culture medium at a concentration equivalent to that of the standard cytokines (molar equivalents, me). The added CCC was IL-2-Fc CCC.

[0196] Figures 11A-11D An example is shown of NK cells imaged under an optical microscope. For example... Figures 11A-11D As shown, donor peripheral blood CD56 on a medium supplemented with CCCs (e.g., IL-2-Fc CCCs and IL-2-Fc CCCs with NK activating factors) + NK cells proliferated significantly more after three days of co-culture than NK cells on culture media supplemented with either a single standard soluble cytokine (e.g., IL-2) or a single Fc cytokine (e.g., Fc-IL-2).

[0197] This disclosure also covers the following provisions: Clause 1. A chimeric cytokine complex comprising: a protein cage polypeptide; a plurality of engineered Fc antibody domains bound to the protein cage polypeptide; and one or more cytokines linked to each of the plurality of engineered Fc antibody domains.

[0198] Clause 2. The chimeric cytokine complex according to Clause 1, wherein the cytokine is an interleukin.

[0199] Clause 3. The chimeric cytokine complex according to Clause 2, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0200] Clause 4. The chimeric cytokine complex according to Clause 1, wherein the plurality of engineered Fc antibody domains comprises six to twelve engineered Fc antibody domains that bind to the protein cage polypeptide.

[0201] Clause 5. The chimeric cytokine complex according to Clause 4, wherein a total of 12 to 24 cytokines are linked to the plurality of engineered Fc antibody domains.

[0202] Clause 6. The chimeric cytokine complex according to Clause 1, wherein each of the one or more cytokines is linked to one of the engineered Fc antibody domains via an engineered metalloproteinase resistance linker sequence.

[0203] Clause 7. The chimeric cytokine complex according to Clause 6, wherein the engineered metalloproteinase resistance linker sequence has a length of between 7 and 13 amino acid residues.

[0204] Clause 8. The chimeric cytokine complex according to Clause 1, wherein the C-terminus of at least one engineered Fc antibody domain is linked to the N-terminus of at least one of the cytokines.

[0205] Clause 9. The chimeric cytokine complex according to Clause 8, wherein one of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0206] Clause 10. The chimeric cytokine complex according to Clause 8, wherein one of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0207] Clause 11. The chimeric cytokine complex according to Clause 8, wherein one of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0208] Clause 12. The chimeric cytokine complex according to Clause 1, wherein the N-terminus of at least one engineered Fc antibody domain is linked to the C-terminus of at least one of the cytokines.

[0209] Clause 13. The chimeric cytokine complex according to Clause 12, wherein one of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0210] Clause 14. The chimeric cytokine complex according to Clause 12, wherein one of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0211] Clause 15. The chimeric cytokine complex according to Clause 12, wherein one of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0212] Clause 16. The chimeric cytokine complex according to Clause 1, wherein the engineered Fc antibody domain is an engineered human Fc antibody domain.

[0213] Clause 17. The chimeric cytokine complex according to Clause 16, wherein the engineered human Fc antibody domain is an engineered human IgG1 Fc antibody domain.

[0214] Clause 18. The chimeric cytokine complex according to Clause 17, wherein one of the engineered human IgG1 Fc antibody domains comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0215] Clause 19. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0216] Clause 20. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0217] Clause 21. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0218] Clause 22. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0219] Clause 23. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0220] Clause 24. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0221] Clause 25. The chimeric cytokine complex according to Clause 18, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0222] Clause 26. The chimeric cytokine complex according to Clause 1, wherein the Fc antibody domain is an engineered rabbit Fc antibody domain.

[0223] Clause 27. The chimeric cytokine complex according to Clause 1 further comprises a signal peptide linked to the N-terminus of at least one engineered Fc antibody domain or at least one cytokine.

[0224] Clause 28. The chimeric cytokine complex according to Clause 27, wherein the signal peptide is a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells.

[0225] Clause 29. The chimeric cytokine complex according to Clause 28, wherein the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0226] Clause 30. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide comprises a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the amino acid sequences shown in SEQ ID NO: 8-15.

[0227] Clause 31. The chimeric cytokine complex according to Clause 30, wherein the amino acid sequence of the polypeptide constituting the protein cage polypeptide includes at least the following point mutation relative to the amino acid sequence shown in any of SEQ ID NO: 8-15: Y294A.

[0228] Clause 32. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16).

[0229] Clause 33. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17).

[0230] Clause 34. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18).

[0231] Clause 35. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19).

[0232] Clause 36. The chimeric cytokine complex according to Clause 1, wherein the protein cage polypeptide self-assembles into a tetrahedral pyramidal structure.

[0233] Clause 37. A method for activating and expanding immune cells, comprising: adding a chimeric cytokine complex to a population of immune cells, wherein the chimeric cytokine complex comprises: a protein cage polypeptide; a plurality of engineered Fc antibody domains bound to the protein cage polypeptide; and one or more cytokines linked to each of the plurality of Fc antibody domains.

[0234] Clause 38. The method described in accordance with Clause 37, wherein the immune cell population is activated and expanded in vitro.

[0235] Clause 39. The method described in accordance with Clause 37, wherein the immune cell population is human donor peripheral blood immune cells.

[0236] Clause 40. The method described in accordance with Clause 37, wherein the immune cell population is live T cells.

[0237] Clause 41. The method described in accordance with Clause 37, wherein the immune cell population is natural killer (NK) cells.

[0238] Clause 42. The method according to Clause 40 further includes activating the T cell population with an anti-CD3 and anti-CD28 T cell activating agent prior to the addition of the chimeric cytokine complex.

[0239] Clause 43. The method according to Clause 37, wherein the cytokine is an interleukin.

[0240] Clause 44. The method according to Clause 43, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7 and IL-15.

[0241] Clause 45. The method according to Clause 37, wherein the plurality of engineered Fc antibody domains comprises six to twelve engineered Fc antibody domains that bind to the protein cage polypeptide.

[0242] Clause 46. The method according to Clause 45, wherein a total of 12 to 24 cytokines are linked to the plurality of engineered Fc antibody domains.

[0243] Clause 47. The method according to Clause 37, wherein each of the one or more cytokines is linked to one of the engineered Fc antibody domains via an engineered metalloproteinase resistance linker sequence.

[0244] Clause 48. The method according to Clause 47, wherein the engineered metalloproteinase-resistant linker sequence has a length of between 7 and 13 amino acid residues.

[0245] Clause 49. The method according to Clause 37, wherein the C-terminus of at least one engineered Fc antibody domain is linked to the N-terminus of at least one of the cytokines.

[0246] Clause 50. The method according to Clause 49, wherein one of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0247] Clause 51. The method according to Clause 49, wherein one of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO:21).

[0248] Clause 52. The method according to Clause 49, wherein one of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0249] Clause 53. The method according to Clause 37, wherein the N-terminus of at least one engineered Fc antibody domain is linked to the C-terminus of at least one cytokine.

[0250] Clause 54. The method according to Clause 53, wherein one of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0251] Clause 55. The method according to Clause 53, wherein one of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0252] Clause 56. The method according to Clause 53, wherein one of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO:25).

[0253] Clause 57. The method according to Clause 37, wherein the engineered Fc antibody domain is an engineered human Fc antibody domain.

[0254] Clause 58. The method according to Clause 57, wherein the engineered human Fc antibody domain is an engineered human IgG1 Fc antibody domain.

[0255] Clause 59. The method according to Clause 58, wherein one of the engineered human IgG1 Fc antibody domains comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0256] Clause 60. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0257] Clause 61. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0258] Clause 62. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0259] Clause 63. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0260] Clause 64. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0261] Clause 65. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0262] Clause 66. The method according to Clause 59, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0263] Clause 67. The method according to Clause 37, wherein the Fc antibody domain is an engineered rabbit Fc antibody domain.

[0264] Clause 68. The method according to Clause 37 further includes a signal peptide linked to the N-terminus of at least one engineered Fc antibody domain or at least one cytokine.

[0265] Clause 69. The method according to Clause 68, wherein the signal peptide is a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells.

[0266] Clause 70. The method according to Clause 69, wherein the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0267] Clause 71. The method according to Clause 37, wherein the protein cage polypeptide comprises a polypeptide having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with any of the amino acid sequences shown in SEQ ID NO: 8-15.

[0268] Clause 72. The method according to Clause 71, wherein the amino acid sequence of the polypeptide constituting the protein cage polypeptide includes at least the following point mutation relative to the amino acid sequence shown in any of the sequences in SEQ ID NO: 8-15: Y294A.

[0269] Clause 73. The method according to Clause 37, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence RWGSGADCAWHLGELVWCTAGSGWE (SEQ ID NO: 16).

[0270] Clause 74. The method according to Clause 37, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence GGRWGADCAWHLGELVWCTAGWEGG (SEQ ID NO: 17).

[0271] Clause 75. The method according to Clause 37, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence GADCAWHLGELVWCTAG (SEQ ID NO: 18).

[0272] Clause 76. The method according to Clause 37, wherein the protein cage polypeptide comprises a polypeptide including a binding site for engineering one of the Fc antibody domains, and wherein the binding site comprises the amino acid sequence RWGSGCDCAWHLGELVWCTCGSGWE (SEQ ID NO: 19).

[0273] Clause 77. The method according to Clause 37, wherein the protein cage polypeptide self-assembles into a tetrahedral pyramidal structure.

[0274] Clause 78. An Fc cytokine complex comprising: an engineered Fc antibody domain; and one or more cytokines linked to said engineered Fc antibody domain.

[0275] Clause 79. The Fc cytokine complex according to Clause 78, wherein the cytokine is an interleukin.

[0276] Clause 80. The Fc cytokine complex according to Clause 79, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

[0277] Clause 81. The Fc cytokine complex according to Clause 78, wherein each of the one or more cytokines is linked to the engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence.

[0278] Clause 82. The Fc cytokine complex according to Clause 81, wherein the engineered metalloproteinase resistance adaptor sequence has a length of between 7 and 13 amino acid residues.

[0279] Clause 83. The Fc cytokine complex according to Clause 78, wherein the C-terminus of the engineered Fc antibody domain is linked to the N-terminus of at least one of the cytokines.

[0280] Clause 84. The Fc cytokine complex according to Clause 83, wherein one of the cytokines is interleukin-2 (IL-2), wherein the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0281] Clause 85. The Fc cytokine complex according to Clause 83, wherein one of the cytokines is interleukin-7 (IL-7), wherein the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0282] Clause 86. The Fc cytokine complex according to Clause 83, wherein one of the cytokines is interleukin-15 (IL-15), wherein the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0283] Clause 87. The Fc cytokine complex according to Clause 78, wherein the N-terminus of the engineered Fc antibody domain is linked to the C-terminus of at least one of the cytokines.

[0284] Clause 88. The Fc cytokine complex according to Clause 87, wherein one of the cytokines is interleukin-2 (IL-2), wherein the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0285] Clause 89. The Fc cytokine complex according to Clause 87, wherein one of the cytokines is interleukin-7 (IL-7), wherein the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0286] Clause 90. The Fc cytokine complex according to Clause 87, wherein one of the cytokines is interleukin-15 (IL-15), wherein the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0287] Clause 91. The Fc cytokine complex according to Clause 78, wherein the engineered Fc antibody domain is an engineered human Fc antibody domain.

[0288] Clause 92. The Fc cytokine complex according to Clause 91, wherein the engineered human Fc antibody domain is an engineered human IgG1 Fc antibody domain.

[0289] Clause 93. The Fc cytokine complex according to Clause 92, wherein the engineered human IgG1 Fc antibody domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0290] Clause 94. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0291] Clause 95. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0292] Clause 96. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0293] Clause 97. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0294] Clause 98. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0295] Clause 99. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0296] Clause 100. The Fc cytokine complex according to Clause 93, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0297] Clause 101. The Fc cytokine complex according to Clause 78, wherein the Fc antibody domain is an engineered rabbit Fc antibody domain.

[0298] Clause 102. The Fc cytokine complex according to Clause 78 further includes a signal peptide linked to the N-terminus of an engineered Fc antibody domain or at least one cytokine.

[0299] Clause 103. The Fc cytokine complex according to Clause 102, wherein the signal peptide is a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells.

[0300] Clause 104. The Fc cytokine complex according to Clause 103, wherein the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0301] Clause 105. A method for activating and expanding immune cells, comprising: adding an Fc cytokine complex to a population of immune cells, wherein the Fc cytokine complex comprises: an Fc antibody domain; and one or more cytokines linked to the Fc antibody domain.

[0302] Clause 106. The method according to Clause 105, wherein the immune cell population is activated and expanded in vitro.

[0303] Clause 107. The method according to Clause 105, wherein the immune cell population is human donor peripheral blood immune cells.

[0304] Clause 108. The method according to Clause 105, wherein the immune cell population is live T cells.

[0305] Clause 109. The method according to Clause 105, wherein the immune cell population is natural killer (NK) cells.

[0306] Clause 110. The method according to Clause 105 further includes activating the T cell population with an anti-CD3 and anti-CD28 T cell activating agent prior to the addition of the Fc cytokine complex.

[0307] Clause 111. The method according to Clause 105, wherein the cytokine is an interleukin.

[0308] Clause 112. The method according to Clause 111, wherein the interleukin is at least one of interleukin-2 (IL-2), IL-7 and IL-15.

[0309] Clause 113. The method according to Clause 105, wherein each of the one or more cytokines is linked to the engineered Fc antibody domain via an engineered metalloproteinase resistance linker sequence.

[0310] Clause 114. The method according to Clause 113, wherein the engineered metalloproteinase-resistant linker sequence has a length of between 7 and 13 amino acid residues.

[0311] Clause 115. The method according to Clause 105, wherein the C-terminus of the engineered Fc antibody domain is linked to the N-terminus of at least one of the cytokines.

[0312] Clause 116. The method according to Clause 115, wherein one of the cytokines is interleukin-2 (IL-2), wherein the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

[0313] Clause 117. The method according to Clause 115, wherein one of the cytokines is interleukin-7 (IL-7), wherein the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

[0314] Clause 118. The method according to Clause 115, wherein one of the cytokines is interleukin-15 (IL-15), wherein the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

[0315] Clause 119. The method according to Clause 105, wherein the N-terminus of the engineered Fc antibody domain is linked to the C-terminus of at least one of the cytokines.

[0316] Clause 120. The method according to Clause 119, wherein one of the cytokines is interleukin-2 (IL-2), wherein the engineered Fc antibody domain is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

[0317] Clause 121. The method according to Clause 119, wherein one of the cytokines is interleukin-7 (IL-7), wherein the engineered Fc antibody domain is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

[0318] Clause 122. The method according to Clause 119, wherein one of the cytokines is interleukin-15 (IL-15), wherein the engineered Fc antibody domain is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

[0319] Clause 123. The method according to Clause 105, wherein the engineered Fc antibody domain is an engineered human Fc antibody domain.

[0320] Clause 124. The method according to Clause 123, wherein the engineered human Fc antibody domain is an engineered human IgG1 Fc antibody domain.

[0321] Clause 125. The method according to Clause 124, wherein the engineered human IgG1 Fc antibody domain comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity with the amino acid sequence shown in SEQ ID NO: 1.

[0322] Clause 126. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and functionally reduce antibody-dependent cytotoxicity (ADCC): P75L, R76W, Y80K, Y80P, Y80R, Y80G, and Y80A.

[0323] Clause 127. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises the following point mutation relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and functionally increase antibody-dependent cytotoxicity (ADCC): Y80W.

[0324] Clause 128. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: S23A, E53A, E77A, Y80F, V87A, A111G, K122A, and D160A.

[0325] Clause 129. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and have a neutral effect on other Fcγ receptors: E117, K118A, and A123T.

[0326] Clause 130. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors and decrease affinity for certain other Fcγ receptors: H52A, R85A, and K106A.

[0327] Clause 131. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to reduce affinity for certain Fcγ receptors: D54A, Q79A, and A111S.

[0328] Clause 132. The method according to Clause 125, wherein the engineered human IgG1 Fc antibody domain comprises at least one of the following point mutations relative to SEQ ID NO: 1 to increase affinity for certain Fcγ receptors: T40A and K74A.

[0329] Clause 133. The method according to Clause 105, wherein the Fc antibody domain is an engineered rabbit Fc antibody domain.

[0330] Clause 134. The method according to Clause 105 further includes a signal peptide linked to the N-terminus of an engineered Fc antibody domain or at least one cytokine.

[0331] Clause 135. The method according to Clause 134, wherein the signal peptide is a mouse Ig heavy chain signal peptide for expression in Chinese hamster ovary (CHO) cells.

[0332] Clause 136. The method according to Clause 135, wherein the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 26).

[0333] Various embodiments have been described. However, those skilled in the art will understand that various changes and modifications can be made to this disclosure without departing from the spirit and scope of the embodiments. Elements of the systems, apparatuses, devices, and methods shown in any embodiment are merely examples of that specific embodiment and can be combined or used in other embodiments of this disclosure. For example, the steps of any method depicted in the drawings or described in this disclosure do not necessarily need to be performed in the specific order or sequence shown or described to achieve the desired result. Additionally, other steps may be provided, or certain steps or operations may be eliminated or omitted from the method or process to achieve the desired result. Furthermore, to achieve the desired result, any component or part of any device or system described in this disclosure or depicted in the drawings may be removed, eliminated, or omitted. Additionally, for the sake of brevity, certain components or parts of the systems, apparatuses, or devices shown or described herein have been omitted.

[0334] Therefore, all other embodiments are within the scope of the following claims, and the description and / or drawings are to be regarded as illustrative rather than restrictive.

[0335] Each of the individual variations or embodiments described and illustrated herein has independent components and features that can be readily separated from or combined with features of any other variation or embodiment. Modifications can be made to adapt particular circumstances, materials, composition, processes, one or more process operations, or one or more steps to one or more objects, spirit, or scope of the invention.

[0336] The method described herein can be executed in any logically possible order of the events, or in the stated order of the events. Furthermore, additional steps or operations can be provided, or certain steps or operations can be eliminated to achieve the desired result.

[0337] Furthermore, if a range of values ​​is provided, every intermediate value between the upper and lower limits of that range, as well as any other value or intermediate value within that range, is covered within the scope of this invention. Additionally, any optional features of the inventive variations may be given and claimed individually or in combination with any or more features described herein. For example, a description of a range of 1 to 5 should be considered as disclosing subranges such as 1 to 3, 1 to 4, 2 to 4, 2 to 5, 3 to 5, etc., and individual digits within that range, such as 1.5, 2.5, etc., and any integers or partial increments therebetween.

[0338] All existing subjects mentioned herein (e.g., publications, patents, patent applications, and journal articles) are incorporated herein by reference in their entirety, except for those parts that conflict with the subject matter of this invention (in which case the content of this document shall prevail). The cited materials are provided solely for their publication prior to the filing date of this application. Nothing herein shall be construed as an admission that this invention is not entitled to precede such materials by prior invention.

[0339] Every reference to a single item includes the possibility that multiple identical items exist. More specifically, unless the context clearly specifies otherwise, the singular forms “a,” “an,” “said,” and “the” as used herein and in the appended claims include plural references. It should be further noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as the premise for the use of such exclusive terms such as “solely,” “only,” etc., or the use of the negative limitation, in relation to the description of the elements of the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0340] Regarding the phrase "at least one of...", when such a phrase modifies multiple items or components (or a list of listed items or components), it refers to any combination of one or more of these items or components. For example, the phrase "at least one of A, B, and C" means: (i) A; (ii) B; (iii) C; (iv) A, B, and C; (v) A and B; (vi) B and C; or (vii) A and C.

[0341] To understand the scope of this disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open-ended terms that specify the presence of a particular feature, element, component, group, integer, and / or step, but do not exclude the presence of other unspecified features, elements, components, groups, integers, and / or steps. The foregoing also applies to words with similar meanings, such as the terms "including," "having," and their derivatives. Furthermore, the terms "part," "section," "section," "component," "element," or "assembly" in their singular form may refer to a single part or a combination of parts. As used herein, the directional terms "forward, backward, upward, downward, vertical, horizontal, below, lateral, sideways, and vertical directions," and any other similar directional terms, refer to those positions of the apparatus or device of the equipment, or the direction in which the apparatus or device of the equipment is translated or moved.

[0342] Finally, as used herein, degree terms such as “approximately,” “about,” and “around” mean a specified value, or a specified value and a reasonable amount of deviation from the specified value (e.g., a deviation of up to ±0.1%, ±1%, ±5%, or ±10%, as the case may be) such that the final result will not be significantly or substantially changed. For example, “about 1.0 cm” could be interpreted as meaning “1.0 cm” or “between 0.9 cm and 1.1 cm.” When degree terms such as “about” or “around” are used to refer to a number or value that is part of a range, the term can be used to modify both the minimum and maximum number or value.

[0343] The structures in the figures may be shown as independent of each other and connected only to a few specific structures, without being connected to other structures. These structures may be combined with each other, perform overlapping functions, or be connected to other structures not shown in the figures. Therefore, the description and / or figures should be considered illustrative rather than restrictive.

[0344] All cited references are incorporated in their full text into this paper by way of citation.

[0345] This disclosure is not intended to be limited to the specific forms given, but rather to cover alternatives, modifications, and equivalents to the variations or implementations described herein. Furthermore, the scope of this disclosure fully encompasses other variations or implementations that may be apparent to those skilled in the art based on the content of this disclosure.

[0346] References [1]Mayes, PA, Hance, KW&Hoos, A. The promise and challenges of immune agonist antibody development in cancer. Nat. Rev. Drug Discov. 17, 509-527 (2018). [2]Spangler, JB, Moraga, I., Mendoza, JL&Garcia, KC Insights into cytokine-receptor interactions from cytokine engineering. Annu. Rev. Immunol. 33, 139-167 (2015). [3]Brooks, AJ et al. Mechanism of activation of protein kinase JAK2 by the growth hormone receptor. Science 344, 1249783 (2014). [4]O’Shea, J.J.&Plenge, R. JAK and STAT signaling molecules inimmunoregulation and immune-mediated disease. Immunity 36, 542-550 (2012). [5]Liao, W., Lin, J.X.&Leonard, W.J. Interleukin-2 at the crossroadsof effector responses, tolerance, and immunotherapy. Immunity 38, 13-25 (2013). [6]Levin, A.M. et al. Exploiting a natural conformational switch toengineer an interleukin-2 ‘superkine’. Nature 484, 529-533 (2012). [7]Powell, M.S.&Hogarth, P.M. Fc receptors. Adv. Exp. Med. Biol. 640,22-34 (2008). [8]de Taeye, S.W. et al. FcgammaR Binding and ADCC Activity of HumanIgG Allotypes. Front. Immunol. 11, 740 (2020). [9]Shields, R.L. et al. High resolution mapping of the binding siteon human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn anddesign of IgG1 variants with improved binding to the Fc gamma R. J. Biol. Chem. 276, 6591-6604 (2001).

[10] Isoda, Y. et al. Importance of the Side Chain at Position 296 ofAntibody Fc in Interactions with FcgammaRIIIa and Other FcgammaReceptors. PLOS One 10, e0140120 (2015).

[11] Edsparr, K., Basse, P.H., Goldfarb, R.H.&Albertsson, P. Matrixmetalloproteinases in cytotoxic lymphocytes impact on tumour infiltration andimmunomodulation. Cancer Microenviron 4, 351-360 (2011).

[12] Benson, H.L. et al. Endogenous matrix metalloproteinases 2 and 9regulate activation of CD4+ and CD8+ T cells. Am J Respir Cell Mol Biol 44,700-708 (2011).

[13] Johnatty, R.N. et al. Cytokine and chemokine regulation ofproMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J. Immunol. 158, 2327-2333 (1997).

[14] de Almeida, L.G.N. et al. Matrix Metalloproteinases: FromMolecular Mechanisms to Physiology, Pathophysiology, andPharmacology. Pharmacol. Rev. 74, 712-768 (2022).

[15] Song, J. et al. PROSPER: an integrated feature-based tool forpredicting protease substrate cleavage sites. PLOS One 7, e50300 (2012).

[16] Liu, X. et al. Human immunoglobulin G hinge regulates agonisticanti-CD40 immunostimulatory and antitumour activities through biophysicalflexibility Nature Communications 10, 4206 (2019)。

Claims

1. A chimeric cytokine complex, comprising: Protein cage polypeptide; Multiple engineered Fc antibody domains that bind to the protein cage polypeptide; as well as One or more cytokines linked to each of the plurality of engineered Fc antibody domains.

2. The chimeric cytokine complex according to claim 1, wherein, The cytokine in question is interleukin.

3. The chimeric cytokine complex according to claim 2, wherein, The interleukin is at least one of interleukin-2 (IL-2), IL-7, and IL-15.

4. The chimeric cytokine complex according to claim 1, wherein, The plurality of engineered Fc antibody domains includes six to twelve engineered Fc antibody domains that bind to the protein cage polypeptide.

5. The chimeric cytokine complex according to claim 4, wherein, A total of 12 to 24 cytokines are linked to the multiple engineered Fc antibody domains.

6. The chimeric cytokine complex according to claim 1, wherein, Each of the one or more cytokines is linked to one of the engineered Fc antibody domains via an engineered metalloproteinase resistance linker sequence.

7. The chimeric cytokine complex according to claim 6, wherein, The engineered metalloproteinase resistance linker sequence has a length of 7 to 13 amino acid residues.

8. The chimeric cytokine complex according to claim 1, wherein, At least one C-terminus of the engineered Fc antibody domain is linked to the N-terminus of at least one of the cytokines.

9. The chimeric cytokine complex according to claim 8, wherein, One of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).

10. The chimeric cytokine complex according to claim 8, wherein, One of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO: 21).

11. The chimeric cytokine complex according to claim 8, wherein, One of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO: 22).

12. The chimeric cytokine complex according to claim 1, wherein, At least one of the N-termini of the engineered Fc antibody domain is linked to the C-terminus of at least one of the cytokines.

13. The chimeric cytokine complex according to claim 12, wherein, One of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to IL-2 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO: 23).

14. The chimeric cytokine complex according to claim 12, wherein, One of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to IL-7 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO: 24).

15. The chimeric cytokine complex according to claim 12, wherein, One of the cytokines is interleukin-15 (IL-15), wherein at least one of the engineered Fc antibody domains is linked to IL-15 via an engineered metalloproteinase resistance linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO: 25).

16. The chimeric cytokine complex according to claim 1, wherein, The engineered Fc antibody domain is an engineered human Fc antibody domain.

17. The chimeric cytokine complex according to claim 16, wherein, The engineered human Fc antibody domain is the engineered human IgG1 Fc antibody domain.

18. A method for activating and expanding immune cells, comprising: A chimeric cytokine complex is added to an immune cell population, wherein the chimeric cytokine complex comprises: Protein cage polypeptide; Multiple engineered Fc antibody domains that bind to the protein cage polypeptide; and One or more cytokines linked to each of the Fc antibody domains.

19. An Fc cytokine complex, comprising: Engineered Fc antibody domain; as well as One or more cytokines linked to the engineered Fc antibody domain.

20. A method for activating and expanding immune cells, comprising: An Fc cytokine complex is added to an immune cell population, wherein the Fc cytokine complex comprises: Fc antibody domain; and One or more cytokines linked to the Fc antibody domain.

Images