Multivalent protein complexes

Abstract

The present disclosure relates to methods for preparing and using multiprotein complexes. The complexes can establish multivalent binding mechanisms for multiple targets in solution or on a surface. The complexes have various applications and can be used, for example, for activation and / or expansion of T cells.

Description

Technical Field

[0001] The present disclosure relates to methods of making and using multivalent protein complexes.

[0002] background

[0003] Creating molecules with multiple binding sites for one or more targets is often difficult. One approach is to chemically conjugate many ligands to polymers. For example, polysaccharides, poly(ethylene glycol) (PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) or synthetic dendrimers are often used for chemical conjugation of binding proteins. However, the preparation of such conjugated molecules is often costly and has low yields, and is difficult to manufacture on a large scale. Another approach is to covalently crosslink the binding ligands with a reactive crosslinker, for example, crosslinking the protein with glutaraldehyde. This crosslinking often produces randomly oriented and clustered protein complexes that often impair their function.

[0004] Since multivalent protein complexes have various applications, there is a need to develop a more efficient method to generate multivalent protein complexes.

[0005] summary

[0006] The present disclosure relates to methods of making and using multivalent protein complexes.

[0007] The present disclosure provides compositions comprising a multiprotein complex comprising a polypeptide having two or more Fc binding domains, and two or more Fc-containing proteins each comprising an Fc region, wherein each Fc-containing protein binds to the Fc binding domain in the polypeptide.

[0008] In some embodiments, the Fc-containing protein is an antibody, a heavy chain antibody, a bispecific antibody, or an Fc fusion protein.

[0009] In some embodiments, a multiprotein complex can bind two or more target molecules in solution or on a solid surface.

[0010] In some embodiments, the Fc binding domain is the IgG binding domain of:

[0011] Staphylococcal protein A or any functional variant thereof, IgG binding domain

[0012] Streptococcal protein G or any functional variant thereof, or the Fc binding domain of a natural or engineered Fc binding protein.

[0013] In some embodiments, two or more Fc binding domains in a polypeptide are connected by a linker sequence.

[0014] In some embodiments, the Fc binding domain has at least

[0015] 90% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0016] In some embodiments, the Fc-containing protein is an Fc-containing protein that binds to one or more of the following: CD2, CD3, CD7, CD11a, CD19, CD26, CD27, CD28, CD30, CD30L, CD40, CD43, CD44, CD45RA, CD46, CD49d, CD62L, CD69, CD81, CD95, CD196, CD127,

[0017] CD137, CD226, BTLA, 0X40, ICOS, GITR, PD1, PDL1, CTLA4, HLA-DR and KLRG-1.

[0018] In some embodiments, two or more multi-protein complexes can be further assembled into supercomplexes through covalent bonds or non-covalent interactions.

[0019] In some embodiments, two or more Fc-containing proteins in the multiprotein complex are identical.

[0020] In some embodiments, the Fc-containing protein is an anti-CD3 antibody. In some embodiments, the Fc-containing protein is an anti-CD28 antibody. In some embodiments, the Fc-containing protein is an antibody that specifically binds to a T cell surface antigen (e.g., CD2, CD27, CD28, CD46, or CD137).

[0021] In some embodiments, the composition comprises a plurality of multiprotein complexes comprising two or more different Fc-containing proteins.

[0022] In some embodiments, the composition comprises a plurality of multiprotein complexes, each multiprotein complex comprising one or more anti-CD3 antibodies and one or more anti-CD28 antibodies.

[0023] Also provided herein are methods for modulating cell function, comprising contacting a cell with a composition described herein.

[0024] Furthermore, the present disclosure describes a method for activating or expanding T cells. The method comprises contacting a T cell with a multiprotein complex comprising a polypeptide having two or more Fc-binding domains and two or more Fc-containing proteins, each comprising an Fc region, wherein each Fc-containing protein binds to an Fc-binding domain in the polypeptide.

[0025] In some embodiments, the multiprotein complex comprises an anti-CD3 antibody and an anti-CD28 antibody.

[0026] In some embodiments, the complex further comprises an anti-CD2 antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD46 antibody, or an anti-CD137 antibody, or a combination thereof.

[0027] Additionally, described herein are methods for activating or expanding T cells. The methods include contacting the T cells with a first multiprotein complex comprising:

[0028] a first polypeptide comprising two or more Fc binding domains and two or more anti-CD3 antibodies, wherein the anti-CD3 antibodies bind to the Fc binding domain in the first polypeptide, and a second multi-protein complex comprising a second polypeptide comprising two or more Fc binding domains and two or more anti-CD28 antibodies, wherein the anti-CD28 antibodies bind to the Fc binding domain in the second polypeptide.

[0029] In some embodiments, the method further comprises contacting the T cell with a third multiprotein complex comprising a third polypeptide comprising two or more Fc binding domains and two or more Fc proteins, each comprising an Fc region, wherein each Fc-containing protein binds to the Fc binding domain in the third polypeptide, wherein the Fc-containing protein is an anti-CD2 antibody, an anti-CD27 antibody, an anti-CD28 antibody, an anti-CD46 antibody, or an anti-CD 137 antibody.

[0030] In some embodiments, the cell is a CD8+ cell or the cell is a CD8+ cell.

[0031] The present disclosure also provides a kit comprising the composition described herein. In some embodiments, the kit comprises a first container comprising

[0032] A composition comprising a polypeptide comprising two or more Fc-binding domains, and a second container comprising a composition comprising a first Fc-containing protein.

[0033] In some embodiments, the kit further comprises a third container comprising a composition comprising a second Fc-containing protein.

[0034] The present disclosure also provides a multi-protein complex comprising a protein comprising two or more Fc binding domains and two or more Fc-containing proteins (e.g., antibodies or Fc fusion proteins). In some embodiments, the Fc-containing protein tightly binds to the Fc binding domain. The complex can be stable and soluble in common buffers and cell culture media, providing a useful tool for cross-linking molecules in solution, on cell surfaces, or on solid surfaces.

[0035] For example, a complex can interact with multiple targets simultaneously. Thus, the complex is a multivalent binding agent of one or more.

[0036] Antibody / Fc fusion proteins can recognize antigens / target molecules, thereby increasing the binding affinity between the complex and the target.

[0037] In some embodiments, the complex is highly flexible due to the inherent flexibility of Fc-binding proteins. The linker region between the Fc-binding domains in the Fc-binding protein can provide the Fc-binding domains with a high degree of freedom, thereby imparting freedom of movement to the bound Fc-containing protein. The flexibility of the complex may be important for its ability to adapt to the shape or contours of the cell surface, thereby providing greater efficiency in recognizing and capturing cell surface targets.

[0038] In some embodiments, the complex is soluble in a buffer and / or culture medium (e.g., cell culture medium). In some embodiments, the complex does not adhere to (e.g., does not bind to) the surface of a plastic, glass, or metal container (e.g., under cell culture conditions).

[0039] In some embodiments, the complex can interact and cross-link multiple copies of a target.The target can be on a surface (eg, a cell surface) or in solution.

[0040] In some embodiments, when the complex comprises two different antibody / Fc fusion proteins that respectively recognize specific cell surface molecules on two cells, the complex can be used to cross-link two targets or two cells.

[0041] In some embodiments, the complex can be used to induce or inhibit certain functions of a cell population in vitro, ex vivo, or in vivo, for example, by cross-linking a plurality of cell surface molecules.

[0042] In some embodiments, the complex can be used for T cell activation and expansion in vitro or ex vivo.

[0043] In one aspect, the present disclosure provides a complex comprising a primary signal for T cell activation and expansion (eg, an anti-CD3 antibody).

[0044] In one aspect, the present disclosure provides a complex comprising a co-stimulatory signal (eg, an anti-CD28 antibody) for T cell activation and expansion.

[0045] In some embodiments, the complex comprises a primary signal and a co-stimulatory signal for T cell activation and expansion. In some embodiments, more than one complex comprising a primary and co-stimulatory signal is used in T cell activation and expansion.

[0046] In some embodiments, the complex may comprise antibodies directed against T cell surface antigens, including, for example, CD2, CD27, CD46, CD137, and / or CD226.

[0047] In some embodiments, the complex can result in efficient expansion of T cells, resulting in a 2- to 100,000-fold expansion in vitro or ex vivo.

[0048] In some embodiments, the expanded T cells may include, for example, CD4+ or CD8+ T cells.

[0049] In some embodiments, the complexes can be used for NK cell activation and proliferation in vitro or ex vivo.

[0050] In some embodiments, the complexes can be used to kill malignant cells, including cancer cells (eg, in vitro or in vivo).

[0051] Furthermore, the present disclosure provides a multi-protein complex comprising an Fc-binding protein and at least two Fc-containing proteins. The complex has the ability to bind and cross-link two or more targets in solution or on a solid surface, wherein the Fc-binding protein is stably associated with the Fc-containing proteins, wherein the Fc-binding protein comprises at least two Fc-binding domains.

[0052] In some embodiments, the Fc binding domain is selected from the group consisting of the immunoglobulin binding domain of Staphylococcal protein A or any functional variant thereof, the IgG binding domain of Streptococcal protein G or any functional variant thereof, and natural or engineered Fc binding proteins / peptides. The Fc binding domain can be connected with or without a linker region.

[0053] In some embodiments, two or more identical Fc-containing molecules bind to one Fc-binding protein to form a monospecific complex.

[0054] In some embodiments, two or more different Fc-containing proteins are conjugated to one Fc-binding protein to form a dual / multispecific complex.

[0055] In some embodiments, two or more complexes can be further assembled into supercomplexes through covalent or non-covalent interactions between Fc binding proteins.

[0056] In some embodiments, the Fc binding protein may comprise additional tags, fragments, domains or modifications in its sequence.

[0057] In some embodiments, the Fc-containing protein comprises an antibody and / or an Fc fusion protein, wherein the antibody is an immunoglobulin molecule (e.g., having two heavy chains and two light chains), a heavy chain antibody, a bispecific antibody, a monoclonal antibody, a polyclonal antibody, or a labeled or modified antibody, wherein the Fc fusion protein is an engineered chimeric protein comprising at least one Fc region.

[0058] In some embodiments, the Fc-containing protein is anti-CD2, anti-CD3, anti-CD7, anti-CD11a, anti-CD19, anti-CD26, anti-CD27, anti-CD28, anti-CD30, CD30L, anti-CD40, anti-CD43, anti-CD44, CD45RA, anti-CD46, anti-CD49d, anti-CD62L, anti-CD69, anti-CD81, anti-CD95, CD196, anti-CD127, anti-CD137, anti-CD226, anti-BTLA, anti-OX40, anti-ICOS, anti-GITR, anti-PD1, anti-PDL1, anti-CTLA4, anti-HLA-DR, anti-KLRG-1 antibody, or a combination thereof.

[0059] In some embodiments, the monospecific complex comprises an anti-CD3 antibody.

[0060] In some embodiments, the monospecific complex comprises an anti-CD28 antibody.

[0061] In some embodiments, the monospecific complex further comprises an antibody against a T cell surface protein, including, for example, CD2, CD27, CD28, CD46, or CD137.

[0062] In some embodiments, the bispecific complex comprises anti-CD3 and anti-CD28 antibodies.

[0063] In some embodiments, the complex has the ability to activate and / or expand T cells in vitro, ex vivo, or in vivo.

[0064] In some embodiments, the complex has the ability to simultaneously target multiple cell surface molecules and / or has the ability to induce or inhibit cellular functions.

[0065] Additionally, the present disclosure describes a kit comprising an Fc-binding protein and at least one Fc-containing protein, wherein the Fc-binding protein and the Fc-containing protein are provided separately, and wherein the Fc-binding protein and the Fc-containing protein can be mixed in a molar ratio to form a complex. In some embodiments, each complex is preformed.

[0066] Also provided in the context of the present invention are methods for inducing or inhibiting a cell function comprising contacting the cell with a plurality of complexes.

[0067] Additionally, described herein are methods for activating and expanding T cells by contacting the T cells with a plurality of complexes described herein. The complexes comprise an anti-CD3 antibody for activating the TCR / CD3 signaling pathway and an anti-CD28 antibody as a co-stimulatory signal.

[0068] In some embodiments, the complex further comprises a co-stimulatory antibody against CD2, CD27, CD28, CD46, or CD137.

[0069] As used herein, the term "protein" refers to a macromolecule having a long chain of one or more amino acid residues. A protein may have one or more polypeptide chains (e.g., 1, 2, 3, 4, 5, 6, or more polypeptide chains). The amino acids in a protein may be modified by other molecules such as sugars and / or lipids.

[0070] As used herein, the terms "multiprotein complex," "protein complex," and "complex" are used interchangeably and refer to a quaternary protein structure having two or more polypeptide chains. The polypeptide chains in a protein complex can be bound to each other by non-covalent interactions or covalent bonds (e.g., disulfide bonds).

[0071] As used herein, the term "supercomplex" refers to a stable association of two or more multiprotein complexes.

[0072] As used herein, the term "domain" refers to a unit of a protein with a unique structure and / or function. A protein domain typically has a recognizable tertiary structure that can exist independently of the rest of the protein.

[0073] As used herein, the term "multidomain" refers to two or more domains in a protein.

[0074] As used herein, the term "Fc" or "Fc region" refers to the "fragment crystallizable" region of an immunoglobulin molecule or variants thereof.

[0075] As used herein, the term "Fc binding protein" refers to a protein that can bind to an Fc region. An Fc binding protein can have 1, 2, 3, 4 or more polypeptides.

[0076] As used herein, the term "Fc binding polypeptide" refers to an engineered or naturally occurring polypeptide that can bind to an Fc region. An Fc binding polypeptide is also an Fc binding protein.

[0077] As used herein, the term "Fc binding domain" refers to a protein domain that can bind to the Fc region.

[0078] As used herein, the term "Fc fusion protein" refers to an engineered chimeric protein having an Fc region and a non-Fc region.

[0079] As used herein, the term "Fc-containing protein" refers to a protein molecule having an Fc region. Fc-containing proteins may include, but are not limited to, antibodies, heavy chain antibodies, antibody variants, and Fc fusion proteins.

[0080] As used herein, the term "multivalent" refers to having more than one antigen or target binding site.

[0081] As used herein, the term "affinity" refers to the enhanced strength of binding between two molecules due to multiple affinities.

[0082] As used herein, the term "crosslinking" refers to the direct or indirect connection of one molecule or target to another molecule or target. Crosslinking can be performed through covalent bonds or non-covalent interactions. In some embodiments, crosslinking is mediated by a crosslinker, which is an intermediate molecule or complex that interacts with the two targets.

[0083] As used herein, the terms "antigen" and "target" are used interchangeably and refer to a molecule that is specifically recognized by an antibody or variant thereof. In some embodiments, the target is a receptor.

[0084] As used herein, the term "Protein A" refers to a 42 kDa surface protein encoded by the gene spa in Staphylococcus aureus. The Ig binding domain of Protein A is an Fc binding domain that has a high affinity for the Fc region of immunoglobulin G.

[0085] As used herein, the terms "domain Z" and "Z domain" refer interchangeably herein to a modified form of domain B of protein A. "Z domain" (SEQ ID NO: 6) comprises a mutation at residue Gly29 of domain B of protein A.

[0086] As used herein, the term "protein G" refers to an immunoglobulin and albumin-binding protein encoded by the gene spg in group C and G Streptococcus bacteria.

[0087] As used herein, the term "linker region" or "linker sequence" refers to a amino acid segment connecting two protein domains. The linker can be an intermediate domain, a loop region, or a disordered region.

[0088] As used herein, the term "affinity tag" refers to a segment of a amino acid sequence that has a specific affinity for another molecule or target. Examples of affinity tags include, for example, poly-Histag, Strep-tag, HA-tag, Flag-tag, GST-tag, and Myc-tag.

[0089] As used herein, the term "monospecific complex" refers to a complex having two or more copies of the same Fc-containing protein (eg, an antibody or Fc fusion protein). A monospecific complex recognizes and binds a single type of antigen / target.

[0090] As used herein, the term "bispecific complex" refers to a complex having two different Fc-containing proteins (e.g., antibodies or Fc fusion proteins). A bispecific complex can recognize and bind to two different targets or two different epitopes on the same target.

[0091] As used herein, the term "multispecific complex" refers to a complex having two or more different Fc-containing proteins (e.g., antibodies or Fc fusion proteins). The multispecific complex can recognize and bind to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different targets or epitopes.

[0092] As used herein, the term "activation" of a T cell refers to the activation of a cell signaling cascade in a T cell that ultimately results in cell proliferation and / or effector function. In some embodiments, activation of a T cell can result in cell death, depending on the primary TCR signal and associated co-stimulatory signals received.

[0093] As used herein, the term T cell "expansion" refers to the proliferation of T cells following activation.

[0094] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe animals, humans or non-humans for whom treatment is provided according to the methods of the present invention. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or minors (e.g., humans under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, micro-animals), pigs, horses, dogs, cats, cattle, and other domestic, farm, and zoo animals.

[0095] As used herein, the term "about" refers to a deviation of + / - 20% of the measured value, where applicable. In some embodiments, the deviation is + / - 10%, + / - 5%, + / - 1% or + / - 0.1% of the measured value.

[0096] 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 belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, the present specification, including definitions, will prevail.

[0097] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] The patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0099] FIG1A is a diagram illustrating an example of a monospecific complex. The complex comprises an Fc-binding protein comprising four Fc-binding domains. Four antibodies bind to the Fc-binding protein via their Fc regions.

[0100] Figure 1B is a diagram showing an example of a bispecific complex having two different types of antibodies (eg, antibodies that specifically bind to different antigens) and a polypeptide comprising four Fc binding domains.

[0101] Figure 2 is a schematic diagram showing the interaction between a bispecific complex and T cell surface markers on T cells. The complex comprises an anti-CD3 antibody (the main signal for T cell activation) and an anti-CD28 antibody (a co-stimulatory signal for T cell activation). The complex can change its conformation and bind more effectively to T cell surface molecules on the surface of T cells.

[0102] Figure 3 is a schematic diagram showing that CD3 is cross-linked by a monospecific complex comprising an anti-CD3 antibody. The cross-linking of CD3 chains leads to the assembly of a CD3-TCR supercomplex. By costimulatory signals (e.g., anti-CD28 antibodies), T cells can be activated and begin to proliferate.

[0103] Figure 4 is a schematic diagram showing cross-linking of two cells via a multiprotein complex. The bispecific complex comprises two different antibodies that can bind to the two cells and bring them into close proximity.

[0104] Figures 5A-5C are a series of images showing that T cells were infected with anti-CD3 antibodies and

[0105] Monospecific complexes containing anti-CD28 antibodies on days 7, 14, and 21. Under static cell culture conditions, T cells expanded by the complexes formed small clusters.

[0106] Figures 5D-5F are a series of images showing T cells expanded by CD3 / CD28-coated magnetic beads ("Dynabeads"). T cells expanded by magnetic beads tend to form larger clusters. The results suggest that the complex can provide better contact between cells and antibodies than magnetic beads.

[0107] Figure 6 Figure 2 is a graph showing the number of T cells expanded by a complex, Dynabeads, ImmunoCult CD3 / CD28 Activator ("TAC"), and Streptamer CD3 / CD28 Premix ("Streptamer"). The complex includes a monospecific anti-CD3 complex and a monospecific anti-CD28 complex, which were mixed in a 1:2 ratio and added to cell cultures. Dynabeads were added to cells at a ratio of approximately 3:1. ImmunoCult TAC and Streptamer Premix were added to cell cultures according to the manufacturer's protocol.

[0108] Figure 7 Figure 2 is a graph showing cell viability on day 21 after expansion with the complex, Dynabeads, TAC, and Streptamer CD3 / CD28 premix, respectively. T cells expanded with the complex had over 99% cell viability, the highest among all tested reagents.

[0109] Figure 8 This graph shows the results of fluorescence-activated cell sorting (FACS), in which T cells were stained with fluorescently labeled anti-CD3, anti-CD4, and anti-CD8 antibodies either after isolation (day 1 after reagent addition) or 13 days after incubation with ComplexEx, Dynabeads, TAC, or Streptamer CD3 / CD28 Premix. When T cells were treated with the complex, the CD8+ population increased by approximately 38%, while the CD4+ population decreased by approximately 12%. The rate of increase in CD8+ cells induced by the complex was much higher than that in the other groups.

[0110] Figure 9 shows the formation and purification of a complex of an Fc binding protein and a human IgG antibody. The Fc binding protein (SEQ ID NO: 10), the IgG antibody and the complex were placed on a gel filtration column (GE).

[0111] Healthcare Superdex S200). The complex was formed by mixing the Fc binding protein and IgG antibody at a molar ratio of approximately 1:3 (as described in Example 2). The elution chromatograms are overlaid to show the differences in elution volumes, which indicate the molecular weights of the three proteins and the stoichiometry of the complex.

[0112] Figure 10 Shown are five exemplary Ig-binding domains of Protein A (SEQ ID NOS: 1-5), the Z domain (SEQ ID NO: 6), the amino acid sequence of the Ig-binding domain of Protein G (SEQ ID NOS: 7-9), and an exemplary Fc-binding protein comprising five tandem Z domains (SEQ ID NO: 10).

[0113] Detailed description

[0114] The present disclosure provides methods for producing and using multivalent protein complexes. Protein complexes can have multiple binding sites that can bind to or interact with multiple target molecules. Such complexes are very useful in situations where molecular interactions (e.g., receptor crosslinking) need to occur simultaneously. For example, the complex can promote the assembly and amplification of CD3-TCR supercomplexes in T cells. Therefore, the complex can accelerate the process of adoptive T cell therapy (ACT).

[0115] Due to its high specificity and long-term immune protection, ACT can be used to treat a variety of human diseases, including cancer. In particular, ACT has become a clinical approach for curative cancer treatment in patients with metastatic disease. In the clinical setting, T lymphocytes are usually expanded in vitro from peripheral blood mononuclear cells (PBMCs), genetically modified T cells, or tumor-infiltrating lymphocytes before infusion. Tumor-specific cytotoxic T cells are then infused into cancer patients with the goal of recognizing, targeting, and destroying tumor cells.

[0116] T cell activation triggers a cell signaling cascade that depends on T cell receptor (TCR) signals and co-stimulatory signals, which can further lead to cell proliferation, effector function or apoptosis. It is well known that T cells require at least two independent signals to be fully activated in vivo (Smith-Garvin et al., "T cell activation." Annual Review of Immunology 27 (2009): 591-619.). In many cases, the primary signal is an antigen-specific signal in which an antigen polypeptide complexed with a major histocompatibility complex (MHC) binds to a T cell receptor (TCR). Co-stimulatory signals are typically mediated by cytokines and / or co-stimulatory signals on antigen presenting cells (APCs). If co-stimulatory signals are not provided, T cells activated only by the primary signal will largely lead to apoptosis.

[0117] Numerous methods have been developed to activate and expand T cells without involving APCs, either in vitro or ex vivo. Studies have shown that TCR complex binding is associated with the assembly of CD3 molecules, which form a supercomplex with the TCR. Crosslinking of the two S chains of CD3 leads to the formation of the TCR-CD3 supercomplex, initiating the signaling cascade that activates T cells. When immobilized on a solid surface, antibodies targeting the CD3 S chain (e.g., the OTK3 clone) can effectively trigger the formation of the TCR-CD3 supercomplex. Furthermore, TCR activation is regulated by several co-stimulatory molecules (e.g., CD28) on the T cell surface. Therefore, immobilized anti-CD3 and anti-CD28 antibodies are commonly used for efficient T cell activation and expansion, either in vitro or ex vivo. For example, magnetic beads coated with anti-CD3 and CD28 antibodies are a popular method for in vitro T cell expansion. It is hypothesized that the surface of the antibody-coated microbeads mimics APCs, leading to rapid T cell proliferation. However, the beaded structure has significant limitations. The rigid surface of the magnetic beads may not be a good substitute for APCs.

[0118] In addition, it may be a challenge to completely remove magnetic beads from T cells (i.e., "de-microsphere") in a clinical setting. Some other similar methods have been developed. However, these methods generally have various limitations and are also difficult to use in a clinical setting. For example, it is shown that tetrameric complexes of CD3 and CD28 are effective for amplifying T cells. Anti-CD3 and anti-CD28 antibodies anchored on streptavidin (tetramers) can also activate / amplify T cells. However, those tetramers may not be very effective in activating T cells due to the rigidity of those antibody complexes. In addition, the high cost of manufacturing those tetramers may make it difficult to use them for large-scale T cell amplification.

[0119] For example, methods for expanding T cells using various reagents are described below, as well as a general description of how T cells may be used in a clinical setting.

[0120] US7572631B2, US2007 / 0036783A1 and US Patent No. US2016 / 0681A; each of which is incorporated herein by reference in its entirety.

[0121] The present invention provides a method for simply, efficiently, and universally forming multiprotein complexes that, when incorporated with a stimulatory signal for T cell activation, can effectively activate / expand T cell populations (e.g., for expanding T cells that can be used in ACT).

[0122] multiprotein complex

[0123] The present disclosure provides a multi-protein complex comprising an Fc-binding protein (e.g., an Fc-binding polypeptide) comprising two or more Fc-binding domains and two or more Fc-containing proteins, each comprising an Fc region, wherein each Fc-containing protein binds to an Fc-binding domain polypeptide in the Fc.

[0124] In some embodiments, the multiprotein complex is assembled from at least one Fc-binding protein and two or more Fc-containing proteins.

[0125] In some embodiments, the Fc binding protein or polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc binding domains. A protein complex can have at least two Fc-containing proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc-containing proteins). Fc-containing proteins can recognize and bind to a specific target. In some embodiments, the protein complex provides more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) binding sites for a specific target.

[0126] In some embodiments, the Fc binding domain in the polypeptide or Fc binding protein is completely occupied by an Fc-containing protein.

[0127] In one embodiment, the Fc binding domain in the polypeptide or Fc binding protein is not fully occupied by the Fc-containing protein. For example, the Fc-containing protein does not occupy 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Fc binding domains.

[0128] In some embodiments, the protein complex can be used to bind multiple copies of a single type of target. In some other embodiments, the protein complex can be used to bind two or more different targets.

[0129] Multi-protein complexes can be multivalent. In some embodiments, complexes can be formed through non-covalent interactions between the Fc binding domain and multiple antibody / Fc fusion proteins.

[0130] In some embodiments, the multiprotein complex is a monospecific complex. A monospecific complex can be formed with an Fc binding protein (e.g., a polypeptide) and one type of antibody / Fc-fusion protein ( FIG. 1A ). The antibody can be a monoclonal antibody or a polyclonal antibody that recognizes one target or molecule. The target or molecule can be in solution or on the surface of a particle (e.g., a nanoparticle). A monospecific complex can bind two or more molecules of the same type.

[0131] In some embodiments, the multiprotein complex is a bispecific complex.

[0132] A bispecific complex can be formed with an Fc-binding protein and two different antibodies / Fc fusion proteins ( FIG. 1B ). The molar ratio of the two antibodies / Fc fusion proteins bound to the Fc-binding protein can vary (e.g., 1:1, 2:1, 3:1, 4:1, 1:2, 1:3, or 1:4). In some embodiments, equal numbers of different antibodies / Fc fusion proteins can bind to a single Fc-binding protein. The bispecific complex can target two different molecules in solution or on the surface of a cell or particle. The bispecific complex can be used to crosslink two different targets (e.g., different molecules, different particles, and / or different cells). For example, as shown in FIG. 2 and FIG. 4 , the bispecific complex can crosslink or aggregate T cells and cancer cells, wherein cell surface markers on T cells and cell surface markers on cancer cells are simultaneously bound in the bispecific complex.

[0133] In some embodiments, the multiprotein complex is a multispecific complex. The multispecific complex can be formed with an Fc binding protein and two or more different antibodies / Fc fusion proteins. The multispecific complex can be used to crosslink multiple different targets in solution.

[0134] Due to the flexibility of Fc-binding proteins, multiprotein complexes exhibit conformational flexibility. For each Fc-binding domain, the interdomain connections can be flexible, allowing for a large degree of freedom. As a result, the complex can better mimic the interaction between antigen-presenting cells and T cells.

[0135] In some embodiments, when the complex is monospecific, the multi-protein complex interacts with one type of cell surface antigen / target. In some other embodiments, when the multi-protein complex is bispecific or multispecific, the complex can interact with more than one cell surface antigen / target. In some embodiments, the complex can be used to crosslink two or more molecules in solution or on a solid surface. In some other embodiments, the complex can be used to crosslink two or more cells or particles.

[0136] In some embodiments, the complexes can be used to modulate cellular functions, such as by cross-linking cell surface molecules to induce or inhibit certain cellular functions.

[0137] In some embodiments, the complexes can be used to activate and expand T cell populations to treat cancer or infectious diseases.

[0138] Fc binding proteins

[0139] The multi-protein complex has a multi-domain Fc binding protein or Fc binding polypeptide that serves as a connection platform for antibodies and their variants (e.g., full-length antibodies, heavy chain antibodies), and Fc fusion proteins. A comprehensive review of common Fc binding proteins can be found in the article "Fc binding ligands for immunoglobulin G: An overview of high-affinity proteins and peptides" (Choe W. et al., Materials. 2016; 9(12)). : 994), which is incorporated herein by reference in its entirety.

[0140] In some embodiments, the Fc-binding protein or polypeptide comprises at least two (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) Fc-binding domains. The Fc-binding domain can be the Ig-binding domain of Staphylococcus protein A or any functional variant thereof, the Z domain or any functional variant thereof, the immunoglobulin-binding domain of Streptococcus protein G or any functional variant thereof, or any other natural or engineered Fc-binding polypeptide. Protein A has five Ig-binding domains, including C (SEQ ID NO: 1), D (SEQ ID NO: 2), E (SEQ ID NO: 3), A (SEQ ID NO: 4), and B (SEQ ID NO: 5). The "Z domain" is a modified B domain (as shown in SEQ ID NO: 6). The Ig-binding domain of protein G from Streptococcus sp. Group G includes C1 (SEQ ID NO: 7), C2 (SEQ ID NO: 8), and C3 (SEQ ID NO: 9). Protein A and Protein G have different affinities for antibodies from different species and / or from different subclasses. Therefore, different Fc binding domains in Fc binding proteins or Fc binding polypeptides can be selected based on the antibody to be used. In some embodiments, the Fc binding domain is a naturally occurring or engineered Fc binding polypeptide, such as the Fc binding domain of Protein A, Protein G, and Protein L. Examples of Fc binding domains or polypeptides can be found in the article "Fc binding ligands for immunoglobulin G: An overview of high-affinity proteins and peptides" (Choe W. et al., Materials. 2016; 9(12): 994), which is incorporated herein by reference in its entirety.

[0141] In some embodiments, the Fc-binding protein of the Fc-binding polypeptide comprises or consists of two Fc-binding domains, three Fc-binding domains, four Fc-binding domains, five Fc-binding domains, six Fc-binding domains, seven Fc-binding domains, eight Fc-binding domains, or more than eight Fc-binding domains.

[0142] Because the Z domain has limited Fab binding activity, Fc-binding proteins or Fc-binding polypeptides having only the Z domain can be used to generate multiprotein complexes. In some embodiments, the Fc-binding protein or Fc-binding polypeptide has five tandemly linked Z domains (SEQ ID NO: 10), which can accommodate 1, 2, 3, 4, or 5 antibodies in solution (e.g., approximately 3-4 antibodies on average in solution). In some embodiments, the antibody is an anti-CD3 or anti-CD28 antibody.

[0143] In some embodiments, the Fc-binding protein or Fc-binding polypeptide has a mixture of Fc-binding domains, such as the C domain, D domain, E domain, A domain and B domain from Protein A, the C2 domain, C2 domain and C3 domain of Protein G, the Z domain, some other Fc-binding domains of Fc-binding polypeptides and variants thereof. Variants of these Fc-binding domains can also be included in the Fc-binding protein or Fc-binding polypeptide. The variants can have amino acid substitutions, deletions and / or additions and still retain the functional activity of the native form of the Fc-binding domain as described herein.

[0144] In some embodiments, the Fc binding domains are connected with or without linker sequences. For example, the five Ig binding domains in wild-type protein A are considered to have no interdomain linker sequences. To increase the flexibility of the Fc binding protein, additional interdomain linker sequences can be added between adjacent domains. Some examples of linker sequences include GGGGGG (SEQ ID NO: 11), GSGSGS (SEQ ID NO: 12), and SSSSS (SEQ ID NO: 13).

[0145] In some embodiments, the Fc binding domain is linked to a chemical linker, such as a disulfide bond, carbodiimide, NHS ester, helylene ester, pentafluorophenyl ester, hydroxymethylphosphine, maleimide, haloacetyl (bromo or iodo), pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazide, alkoxyamine, diazo, aryl azide, or isocyanate.

[0146] Each Fc binding domain can have a high affinity for the Fc region of an antibody / Fc fusion protein. Thus, each Fc binding protein can accommodate two or more copies of an antibody / Fc fusion protein or two or more molecules of Fc / fusion protein.

[0147] Different antibody / Fc fusion proteins. In some embodiments, the complex provides a high local ligand density for binding to the antigen / target, resulting in enhanced binding affinity.

[0148] In some embodiments, the Fc-binding protein or polypeptide comprises two or more copies of a single Fc-binding domain (eg, a Z domain).

[0149] In some embodiments, an Fc-binding protein or polypeptide comprises two or more different Fc-binding domains (eg, a B domain and a Z domain).

[0150] In some embodiments, the Fc-binding polypeptide is engineered to include one or more cysteine ​​residues for further cross-linking of the Fc-binding polypeptide via disulfide bonds.

[0151] In some embodiments, the Fc binding protein or polypeptide is engineered to include some additional functional domains or sequences.

[0152] In some embodiments, the Fc-binding protein or Fc-binding polypeptide includes an affinity tag, such as, but not limited to, a poly-His tag, a Strep-tag, an HA-tag, a Flag-tag, or a GST-tag. The affinity tag, if included, can provide a purification tool for the multi-protein complex. The affinity tag can also provide a method for immobilizing the multi-protein complex on a surface or another molecule.

[0153] In some embodiments, the Fc-binding protein, whether wild-type or engineered, can be synthesized in expression systems such as bacteria (e.g., E. coli), yeast, algae, insect cells, and mammalian cells. After the Fc-binding protein is purified to homogeneity, the Fc-binding protein is quantified and reconstituted in an appropriate buffer to the desired concentration.

[0154] In some embodiments, the Fc binding domain can have a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to the immunoglobulin binding domains described in the present disclosure (e.g., domains B, C, A, E, D, Z, C1, C2, and C3).

[0155] In some embodiments, the Fc binding polypeptide may have a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.

[0156] Fc-containing proteins

[0157] An Fc-containing protein is a protein molecule that has an Fc region. Fc-containing proteins can include, for example, antibodies, heavy-chain antibodies, and Fc-fusion proteins. Fc-fusion proteins can be engineered chimeric proteins that contain at least one Fc region. Fc-containing proteins that contain an Fc region can associate with Fc-binding proteins via the Fc region.

[0158] The antibody can be a monoclonal antibody, a polyclonal antibody or a variant thereof. The antibody can also be an immunoglobulin molecule (comprising two heavy chains and two light chains), or a two-chain heavy chain antibody.

[0159] The Fc-containing protein can have specific binding affinity for an antigen or target in solution or on a solid surface. In some embodiments, the Fc-containing protein is an antibody or Fc fusion protein that can bind to a cell surface marker. In some embodiments, the cell surface marker is a tumor cell surface marker.

[0160] In some embodiments, the antibody or variant thereof is derived from a single animal source or from multiple animal sources, including but not limited to mice, rats, rabbits, horses, sheep, goats, cows, camelids, sharks, pigs, hamsters, humans. In some embodiments, the antibody is extracted from an animal. In other

[0161] Embodiment, purify antibody / Fc fusion protein from recombinant protein expression system.

[0162] In some embodiments, the Fc-containing protein is an antibody that can recognize a T cell surface marker. In some embodiments, the Fc-containing protein provides a primary signal (e.g., anti-CD3) and / or a co-stimulatory signal (e.g., anti-CD28, anti-CD2, anti-CD27, anti-CD46) for T cell activation and expansion. (Anti-CD137, anti-CD226) is used for T cell activation and expansion. In some embodiments, the Fc-containing protein can specifically bind to CD3, CD28, CD2, CD27, CD46, CD137, or CD226.

[0163] In some embodiments, the Fc-containing protein is a bispecific antibody. It can provide primary and costimulatory signals for T cell activation and expansion. In some embodiments, the first arm of the bispecific antibody can bind to CD3, and the second arm of the bispecific antibody can bind to CD28, CD2, CD27, CD46, CD137, or CD226.

[0164] Methods for preparing multiprotein complexes

[0165] The complexes can be prepared in a variety of different ways, and complexes of Fc-binding proteins and antibody / Fc-fusion proteins can be formed with varying titers.

[0166] To prepare a monospecific complex, the selected antibody / Fc fusion protein can be mixed with a purified Fc-binding protein at a certain molar ratio. Although more molar amounts of antibody / Fc-fusion protein are used for mixing, the antibody / Fc-binding protein may or may not saturate the binding sites on the Fc-binding protein due to steric hindrance in the Fc-binding protein. In some embodiments, the molar ratio of Fc-binding protein to Fc-containing protein is about 1:2 to about 1:100 (e.g., about 1:2 to about 1:10, or between about 1:2 and about 1:5).

[0167] To produce a bispecific or multispecific complex, equimolar amounts of two or more antibodies / Fc-fusion proteins can be first mixed. The mixture of antibodies / Fc fusion proteins is further mixed with an Fc binding protein or Fc binding polypeptide at a desired molar ratio. Alternatively, a desired molar ratio of two or more antibodies / Fc fusion proteins is mixed. The resulting mixture is further mixed with an Fc binding protein at a desired molar ratio. In some embodiments, the molar ratio of the two different antibodies / Fc fusion proteins used to form the bispecific complex can be between about 1:1 and about 1:5 (e.g., about 1:1, 1:2).

[0168] 1:3, 1:4, or 1:5). In some embodiments, the various antibodies / Fc fusion proteins used to form the multispecific complex are in approximately equal moles.

[0169] The interaction of the mixed components may be rapid. Thus, a complex may be formed in a short period of time (e.g., less than 6 hours, 1 hour, 10 minutes, or 1 minute).

[0170] The complex can be further purified by chromatography. Complex purification can be based on affinity tags attached to Fc-binding proteins. Ion exchange chromatography can be used to separate the various complexes in solution. Ion exchange chromatography can separate different populations of complexes based on the differential charge of the various antibodies / Fc-fusion proteins in the complex.

[0171] In some embodiments, the complex is stable and soluble within a certain temperature range, for example, 0-40°C, preferably 4-37°C.

[0172] In some embodiments, two or more compounds are mixed together to form a composition.

[0173] In some embodiments, two or more complexes are mixed together and further cross-linked using disulfide bonds or other chemical linkers.

[0174] In some embodiments, after the multi-protein complex is generated, it can be further purified using liquid chromatography, such as, but not limited to, size exclusion / gel filtration, affinity chromatography, ion exchange chromatography, and hydrophobic chromatography. The purified complex in solution can be concentrated or diluted to a desired concentration. In some embodiments, the complex in solution can be stored for extended periods of time under various conditions, such as at different temperatures.

[0175] The present disclosure further provides vectors comprising sequences encoding amino acid sequences as described herein and cells comprising these vectors. The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising nucleic acids disclosed herein (e.g., nucleic acids encoding polypeptides disclosed herein), introducing recombinant vectors into host cells (i.e., making the host cell comprise polynucleotides and / or vectors comprising polynucleotides), and producing polypeptides or their proteins by recombinant techniques.

[0176] The present disclosure also provides for nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein and have an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

[0177] are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any polypeptide described herein, or any amino acid sequence encoded by any nucleotide sequence described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40,

[0178] In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, or 500 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 amino acid residues.

[0179] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any sequence described herein. In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence

[0180] sequence, wherein the nucleic acid sequence is any sequence described herein.

[0181] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acids or nucleic acids). For comparison purposes, amino acid sequences and non-homologous sequences used for optimal alignment can be disregarded). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which needs to be introduced to achieve optimal alignment of the two sequences. For purposes of the present invention, the comparison of two sequences and determination of percent identity between the two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0182] Methods of using the compound

[0183] The multiprotein complex can be used to bind to a variety of cell surface proteins to regulate cell functions, such as inducing or inhibiting cell functions.

[0184] In some embodiments, the complex can bind to a population of T cells, providing primary and costimulatory signals to the T cells. In some embodiments, the complex can be used to activate and / or expand T cells.

[0185] Initial T cells can be obtained from peripheral blood mononuclear cells

[0186] (PBMC) blood of a donor of a subject (e.g., a human). T cells can also be obtained from many other sources, including bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any T cell line known in the art can be used. The T cells used in this experiment can be isolated by consuming B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and red blood cells. The technology of T cell isolation is known to those of ordinary skill in the art.

[0187] In some embodiments, a monospecific complex comprising two or more copies of an anti-CD3 antibody is provided as the primary signal for T cell activation and proliferation. The monospecific anti-CD3 complex cross-links the S chain of CD3 and triggers the assembly of the CD3-TCR supercomplex on T cells ( FIG. 3 ). The assembled CD3-TCR transduces a single cascade reaction, thereby activating T cells.

[0188] In some embodiments, monospecific complexes comprising two or more copies of an anti-CD28 antibody can provide a co-stimulatory signal for T cell activation.

[0189] In some embodiments, a monospecific complex comprising two or more copies of an antibody selected from, but not limited to, anti-CD2, anti-CD3, anti-CD27, anti-CD46, anti-CD 137, and anti-CD226 can be used as another co-stimulatory signal.

[0190] In some embodiments, complexes comprising a primary signal and a stimulatory signal are used to treat T cells. For example, a monospecific anti-CD3 complex and a monospecific anti-CD28 complex can be used together for effective T cell activation and expansion.

[0191] In some embodiments, the molar ratio of different monospecific complexes used to treat T cells is adjustable. For example, the molar ratio of the complex with the primary signal (e.g., anti-CD3) to the complex with the costimulatory signal (e.g., anti-CD28) can be between about 1:1 and about 1:100. The molar ratio of the complex with the primary signal (e.g., anti-CD3) to the complex with the costimulatory signal (e.g., anti-CD28) can also be between about 1:100 and about 1:1 ( FIG. 2 ).

[0192] The ratios described herein (e.g., primary signal:costimulatory signal, or costimulatory signal:primary signal) can be greater than 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:2, or 1:1. The ratios can also be less than 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:2, or 1:1.

[0193] In some embodiments, uncomplexed antibodies and monospecific complexes comprising anti-CD3 antibodies providing costimulatory signals are used together for T cell activation (Fig. 3). In some embodiments, anti-CD28 antibodies and monospecific complexes comprising anti-CD3 are used together for T cell activation. In some other embodiments, two or more uncomplexed costimulatory antibodies and monospecific complexes comprising anti-CD3 are used for T cell activation. As used herein, the term "uncomplexed" refers to soluble antibodies that are not associated with Fc binding proteins.

[0194] In some embodiments, the additional co-stimulatory signal comprises an antibody selected from, but not limited to, anti-CD2, anti-CD7, anti-CD27, anti-CD46, anti-CD 137, and anti-CD226. These antibodies can be complexed or uncomplexed and added to the monospecific complex of anti-CD3 and anti-CD28 to activate T cells.

[0195] In some embodiments, bispecific complexes comprising both anti-CD3 and anti-CD28 antibodies are used for T cell activation ( FIG. 2 ).

[0196] In some embodiments, bispecific complexes comprising anti-CD3 and an antibody for a co-stimulatory signal selected from, but not limited to, anti-CD2, anti-CD7, anti-CD27, anti-CD46, anti-CD137, and anti-CD226 can be used for T cell activation.

[0197] In some embodiments, the additional co-stimulatory signal comprises complexed and / or non-complexed antibodies selected from, but not limited to, anti-CD2, anti-CD7, anti-CD27, anti-CD46, anti-CD137, and anti-CD226. Anti-CD3 and anti-CD28 dual-specific complexes can be added to activate T cells.

[0198] In some embodiments, the different preassembled complexes and any uncomplexed antibodies are added separately to the T cell culture medium. In some other embodiments, the different preassembled complexes and any uncomplexed antibodies are premixed before being added to the T cell culture medium.

[0199] In some embodiments, the cell culture vessel is blocked with a non-toxic hydrophilic material to prevent potential immobilization of the multi-protein complex. At least a portion of the complex remains soluble in the cell culture medium.

[0200] In some embodiments, each T cell to be activated is covered with at least one complex comprising anti-CD3 and one complex comprising anti-CD28 based on calculation of the molar concentrations of the complexes. Additional complexes comprising anti-CD3 and / or anti-CD28 can be added during the expansion of the T cells.

[0201] In some embodiments, excess complexes comprising anti-CD3 and / or anti-CD28 are removed by an affinity resin that specifically binds to the complex (eg, an affinity tag in the complex).

[0202] In some embodiments, T cells activated by the complexes described herein can be expanded by about 2-100,000 fold (e.g., more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 fold), 40, 50, 60, 70, 80, 90, 100, 500, 1000, 5000, 10000, 50000, or 100,000 fold) within 2 to 30 days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 days). In some embodiments, T cells can be expanded by more than 10 fold, 100 fold, 1000 fold, 2000 fold, 4000 fold,

[0203] By using the multiprotein complexes disclosed herein, increases of 100,000-fold or more can be achieved over a suitable period of time (eg, 21 days).

[0204] In some embodiments, the complex is very effective for in vitro and ex vivo T cell expansion. In some embodiments, the efficiency of T cell expansion by the methods described herein is about 2-20 times higher than magnetic bead-based expansion.

[0205] In some embodiments, T cells expanded by the complex have equal or fewer terminally amplified cells or apoptotic cells compared to bead-based expansion.

[0206] In some embodiments, T cells expanded by the complexes described herein have greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% cell viability (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days after initiation of expansion).

[0207] In some embodiments, the T cells expanded by the complex are in a ratio of 1: 1 to 1: 20 (e.g., 1: 2, 1: 3, 1: 4, 1: 5, 1: 10, 1: 15, or 1: 20). In some embodiments, the complexes and methods described herein can induce a T cell population with a larger proportion of CD8+ cells compared to bead-based expansion. In some embodiments, the ratio of CD8+ cells in T cells (e.g., CD8+ cells and CD4+ cells) is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the ratio of CD4+ cells in T cells (e.g., CD8+ cells and CD4+ cells) is less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the complexes described herein can increase the percentage of CD8+ cells by more than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some embodiments, the complexes described herein can decrease the percentage of CD4+ cells in T cells by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0208] In some embodiments, T cells are collected from a subject and expanded by the methods described herein. The expanded T cells can be used for a variety of purposes, such as adoptive cell transfer.

[0209] In some embodiments, bispecific complexes comprising two different antibodies / Fc fusion proteins can be used to crosslink two different cells by having one cell bind to one antibody / Fc fusion protein on the complex and bind to the other cell that is bound to the other antibody / Fc fusion protein in the complex.

[0210] In some embodiments, T cells are expanded in the presence of IL-2.

[0211] In some embodiments, a bispecific complex comprising an antibody that recognizes an antigen on a T cell and another antibody that recognizes a tumor cell surface marker can crosslink the T cell to the tumor cell ( FIG. 4 ). In some embodiments, crosslinking can kill the tumor cell.

[0212] Therefore, the present disclosure also provides a method for killing cancer cells. The method may include contacting cancer cells with a multiprotein complex as described herein. In some embodiments, the multiprotein complex can recognize and bind to specific cell surface molecules on immune cells (e.g., CD2, CD27, CD46, CD 137 and / or CD226), as well as specific cell surface molecules on cancer cells (e.g., PD-L1, Her2 and CD20). Therefore, the present disclosure also provides a method for treating cancer in a subject.

[0213] In some embodiments, the complex can be used to capture and isolate a specific type of cell. The complex can include an antibody / Fc fusion protein that recognizes a specific surface protein on the cell to be captured.

[0214] In one embodiment, the Fc binding protein may have an affinity tag, including, for example, a poly-His tag and a Strep tag. Once the cells are bound by the various complexes, a matrix comprising solid particles or a membrane and a binding moiety that specifically interacts with the affinity tag on the Fc binding protein in the complex can be used to pull down the cells.

[0215] Ingredients and kits

[0216] The present disclosure also provides a composition or a pharmaceutical composition comprising the complex.

[0217] In some embodiments, the composition is mixed with a pharmaceutically acceptable carrier.The pharmaceutical compositions and formulations can be used in vitro, in vivo or ex vivo, or can be administered parenterally, topically, orally or topically.

[0218] In some embodiments, due to the high affinity interaction between the Fc-binding protein and the protein binder, the complex is stable in a buffer (e.g., phosphate buffer, bicarbonate buffer) or a medium (e.g., DMEM, X-VIVO-15). Antibody / Fc-fusion protein. The complex can maintain its structure under various conditions, such as various temperatures (e.g., 0°C to 37°C), various pH values ​​(e.g., pH 5 to pH 9), various ionic strengths (e.g., 0-2,000 mS / cm)), and / or in the presence of a detergent (e.g., TWEEN-20).

[0219] In some embodiments, the complex is completely soluble in a solution, buffer, or medium over a wide range of temperature (eg, 4°C to 37°C) and pH (eg, pH 5 to pH 9).

[0220] The present disclosure further provides a T cell expansion kit comprising a multi-protein complex, antibodies, other components, and instructions for use thereof.

[0221] Implementation Examples

[0222] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0223] Example 1: Materials and methods

[0224] The following materials and methods were used in the examples.

[0225] Source of T cells

[0226] Prior to expansion, starting T cells were obtained from peripheral blood mononuclear cells (PBMCs) of donor human blood. The T cells used in the examples were isolated by depletion of B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes, and erythrocytes.

[0227] T cell culture

[0228] First, a 48-well cell culture plate was blocked with phosphate-buffered saline (PBS) containing 2.0% human serum albumin (HSA) for 18 hours at 37°C. Blocking with HSA prevents the fixation of multiprotein complexes on the plastic surface. Excess HSA was washed away with PBS. T cells isolated from PBMC were isolated in X-VIVO TM 15 (Lonza, Basel, Switzerland, London) with 5% human AB serum and 10-20 IU / mL IL-2, with an initial cell density of 0.1-1.0×106 / mL. All cells were cultured in an incubator at 37°C, 5% CC, and 75% humidity.

[0229] Antibody

[0230] Mouse anti-human CD3 ("OKT3") and mouse anti-human CD28 ("CD28.2") monoclonal antibodies were purchased from Life Technologies (Carlsbad, CA). Both antibody solutions were diluted to 0.2 pg / pL.

[0231] Fc binding proteins

[0232] A polypeptide containing five Z domains (SEQ ID NO: 10) was designed and expressed in a yeast system. The secreted protein was purified using a Superdex S75 gel filtration column (GE Healthcare, Boston, MA). The purified protein was reconstituted in PBS to a concentration of 0.2 pg / pL.

[0233] magnetic beads

[0234] Dynabeads ClinExVivo™ CD3 / CD28 (or "Dynabeads", purchased from Thermo Fisher Scientific, Waltham, MA) were used for comparison purposes. A 3:1 beads to cell ratio was used in the experiments.

[0235] Tetrameric Antibody Complex (TAC)

[0236] T cell activator "ImmunoCult™ Human CD3 / CD28 T Cell Activator" (from STEMCELL Technologies, Vancouver, Canada) was also used for comparison purposes. Approximately 25 μL of TAC solution was added to 1 mL of culture medium to expand T cells.

[0237] Streptamer CD3 / CD28 premix

[0238] T cell activator Streptamer CD3 / CD28 premix solution (from IBA Lifesciences, Göttingen, Germany) was also used for T cell expansion.

[0239] Example 2: Formation of multiprotein complexes

[0240] Experiments were performed to prepare monospecific complexes of anti-CD3 and anti-CD28 antibodies. To confirm the formation of complexes between Fc-binding proteins and human IgG antibodies and to assess their stoichiometry, the complexes, Fc-binding proteins, and human IgG were subjected to ultra-strong gel filtration chromatography (GE Healthcare).

[0241] S200) (Figure 9). The molecular weight of each population was estimated based on the elution volume. Based on titration and size exclusion experiments, each Fc-binding protein can bind approximately three IgG molecules. Therefore, a molar ratio of Fc-binding protein to antibody of 1:3 was used to mix the Fc-binding protein and antibody.

[0242] Briefly, 90 pg of anti-CD3 antibody or anti-CD28 antibody was mixed with 10 pg of Fc binding protein. The mixed solution was sterile filtered and stored at 4 ° C. Optionally, the monospecific complex formed was purified using a Superdex 200 column (GE Healthcare, Boston, MA). The elution peak of the complex was completely separated from the peak of the Fc binding protein or antibody. The collected complex fractions were quantified using a BCA protein assay kit (purchased from Thermo Fisher Scientific, Waltham, MA) and the protein concentration was adjusted to 100-200 pg / ml.

[0243] Bispecific complexes containing both anti-CD3 and anti-CD28 are also prepared by mixing Fc-binding protein with anti-CD3 and anti-CD28 antibodies. First, equimolar concentrations of anti-CD3 and anti-CD28 are mixed. The antibody mixture is then mixed with the Fc-binding protein. This results in a 50% bispecific complex, a 25% monospecific anti-CD3 complex, and a 25% monospecific anti-CD28 complex. Briefly, 45 μg of anti-CD3 antibody and 45 μg of anti-CD28 antibody are thoroughly mixed. Approximately 10 μl of Fc-binding protein is then added to the mixture. This complex solution is used to treat T cells.

[0244] Example 3: Expansion of T cells

[0245] Because T cells typically increase in number tens or hundreds of times during in vitro / ex vivo expansion, they are periodically split when they are approximately 70-90% confluent. Splits are performed at a ratio of approximately 1:2 to 1:5. The culture medium is replenished after cell splitting. Additional T cell expansion agents can be introduced into subcultures to compensate for dilution caused by splitting.

[0246] Briefly, T cells isolated from PBMCs were collected, counted, and evenly distributed into the wells of a 48-well plate previously blocked with HSA. Each well contained a starting number of 0.1 x 10 cells in a volume of 100 μL.

[0247] In one experiment, wells were grouped according to stimulation method.

[0248] Each group had three replicate wells, treated equally. 3 μL of PBS was added to Group 1 (negative control). Group 2 was treated with 2 μL of anti-CD3 complex and 3 μL of anti-CD28 complex. Group 3 was treated with 3 μL of CTS Dynabeads CD3 / CD28 (ThermoFisher) at a cell to bead ratio of approximately 1:3. Group 4 was treated with 3 μL of ImmunoScan CD3 / CD28 (Stemcell Technologies Inc). Group 5 was treated with 5 μL of CD3 / CD28 Streptomyces premix (IBA Lifesciences).

[0249] Subculture T cells for 2 days in the presence of expansion medium or PBS before changing the medium. Add fresh liquid T cell expansion medium immediately after changing the medium. Keep Dynabeads CD3 / CD28 attached to the cells during the medium change. Count cells every two days after splitting.

[0250] Results: As shown in the figure. As shown in Figure 6, Group 2 treated with a multiprotein complex containing CD3 / CD28 showed higher T cell expansion efficiency. For comparison, the efficiency of the T cell expansion complex was approximately 50% to 1,000% higher than other expanders including Dynabeads CD3 / CD28, Immunocult CD3 / CD28 ("TAC"), and CD3 / CD28 Streptamers premix ("Streptamer"). The starting population of T cells was approximately 0.1×106, and the cells were cultured in the presence of approximately 10 IU of IL-2. The cells were split and counted at intervals of 1-3. The cells treated with the complex had a higher expansion rate than the other tested reagents.

[0251] Table 1. Expansion multiples using different expansion agents

[0252]

[0253] Example 4: Viability of cells expanded by different expansion agents

[0254] After 21 days of expansion, T cells were stained with propidium iodide for viability testing, as shown in Figure 1. As shown in Figure 7, the viabilities of cells expanded with the complex, Dynabeads CD3 / CD28, ImmunoCult CD3 / CD28 Activator, and Streptamer CD3 / CD28 Master Mix were 99.2%, 94.1%, 94.5%, and 88.3%, respectively. Therefore, the protein complex does not induce significant T cell death after activation and expansion. In contrast, T cells depleted with the other expansion agents experienced approximately 5-12% cell death.

[0255] Example 5: CD4+ and CD8+ population analysis

[0256] The following monoclonal antibodies conjugated with fluorescent dyes were used for flow cytometric analysis: FITC-conjugated anti-CD4, anti-CD8 (Dako Cytomation, Denmark), anti-CD3 (BD ​​Biosciences, San Jose, CA) antibodies; PE-coupled anti-CD4 antibody; PE-Cy-5-conjugated anti-CD8 antibody (Dako Cytomation, Denmark).

[0257] T cells were stained with FITC-conjugated or phycoerythrin (PE)-conjugated antibodies, including monoclonal mouse anti-human CD4 and CD8, polyclonal goat anti-mouse antibodies (Sigma, St. Louis, MO), and monoclonal mouse anti-human IgG, Fc fragment-specific F(ab)2 antibodies (Jackson ImmunoResearch, West Grove, PA) for analysis of cell surface immunophenotype.

[0258] The cells were washed and resuspended in 50 μL of Hanks buffered saline solution containing 2% FBS and 5 μL of stock antibody preparation. After incubation at 4-10°C for 10 minutes, the cells were washed twice, resuspended in 300 μL of PBS containing 1% paraformaldehyde, and analyzed using a FACScan (BD Biosciences Immunocytometry Systems, San Jose, CA).

[0259] For each group, greater than 96% of T cells expressed CD3. As shown in Figure 4, the ratio of CD4+ to CD8+ cells varied with the different expanders. Table 8 summarizes the results.

[0260] Table 2. Relative changes in CD4+ and CD8+ populations

[0261]

[0262] T cells expanded with the protein complex had a significantly higher CD8+ subset than those expanded with the other expanders. This suggests that the protein complex can stimulate the CD8+ subset. Interestingly, T cells expanded with the complex, TAC, and Streptamer all had a higher CD8+ subset and a lower CD4+ subset than cells expanded with Dynabeads.

[0263] Example 6: Functional characteristics of expanded T cells

[0264] T cells from PBMCs were activated and expanded in the presence of two monomeric complexes (i.e., anti-CD3 complex and anti-CD28 complex) or Dynabeads CD3 / CD28. Briefly, approximately 100 pL / well of T cells at a starting concentration of 0.1×106 / mL were incubated with 100 IU / mL of IL-2. Approximately 2 pL of anti-CD3 monomeric complex and approximately 3 pL of anti-CD28 monomeric complex were added to the cells. Dynabeads CD3 / CD28 Expander was used for comparison purposes.

[0265] Microspheres were added to cells at a final microsphere to cell ratio of 3: 1. For negative controls, blank PBS was added to the wells.

[0266] After 13 days of expansion, cells were harvested, washed, and plated into new wells without IL-2. Expansion agent or PBS was added. After 24 hours of growth, cell culture supernatants were collected for analysis of secreted cytokines.

[0267] Levels of IL-2, IL-4, INFγ, and TNFα were measured by enzyme-linked immunosorbent assay (ELISA). The pattern of cytokine secretion by T cells depends on many factors, including cell subsets and stimulation conditions. Protein complexes and magnetic bead expansion agents result in slightly different T cell subsets. Furthermore, different crosslinking of CD3 / CD28 molecules may trigger distinct activation signals within T cells. It is expected that cytokine levels will differ between T cells activated and expanded using protein complexes and Dynabeads.

[0268] Other implementations

[0269] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims. Sequence Listing <110> Bioprosea Technologies LLC <120> Multivalent protein complexes <130> 44604-0003WO1 <140> PCT / US2019 / 033079 <141> 2019-05-20 <150> US 62 / 674,375 <151> 2018-05-21 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 1 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 2 <211> 60 <212> PRT <213> Staphylococcus aureus <400> 2 Ala Ala Asn Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr 1 5 10 15 Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe 20 25 30 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly 35 40 45 Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys 50 55 60 <210> 3 <211> 61 <212> PRT <213> Staphylococcus aureus <400> 3 Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe 1 5 10 15 Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu 35 40 45 Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 50 55 60 <210> 4 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 4 Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ser Glu Ala 35 40 45 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 50 55 <210> 5 <211> 58 <212> PRT <213> Staphylococcus aureus <400> 5 Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 6 <211> 58 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Z domain (B domain of Protein A with mutations) <400> 6 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 7 <211> 55 <212> PRT <213> Streptococcus sp. <400> 7 Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr 1 5 10 15 Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr 20 25 30 Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr 35 40 45 Lys Thr Phe Thr Val Thr Glu 50 55 <210> 8 <211> 55 <212> PRT <213> Streptococcus sp. <400> 8 Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr 1 5 10 15 Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr 20 25 30 Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr 35 40 45 Lys Thr Phe Thr Val Thr Glu 50 55 <210> 9 <211> 55 <212> PRT <213> Streptococcus sp. <400> 9 Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr 1 5 10 15 Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr 20 25 30 Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr 35 40 45 Lys Thr Phe Thr Val Thr Glu 50 55 <210> 10 <211> 298 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Five tandemly connected Z domains <400> 10 Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile 1 5 10 15 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn 50 55 60 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu 65 70 75 80 Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro 85 90 95 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala 100 105 110 Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala 115 120 125 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn 130 135 140 Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu 145 150 155 160 Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Val Asp 165 170 175 Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His 180 185 190 Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu 195 200 205 Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys 210 215 220 Leu Asn Asp Ala Gln Ala Pro Lys Val Asp Asn Lys Phe Asn Lys Glu 225 230 235 240 Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu 245 250 255 Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln 260 265 270 Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala 275 280 285 Pro Lys His His His His His Cys 290 295 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Linker sequences <400> 11 Gly Gly Gly Gly Gly Gly 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Linker sequences <400> 12 Gly Ser Gly Ser Gly Ser 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Linker sequences <400> 13 Ser Ser Ser Ser Ser 1 5

Claims

1. A method for regulating cell function, characterized in that The method comprises: (a) generating a multi-protein complex by mixing a polypeptide with two or more Fc-containing proteins, wherein the multi-protein complex contains two or more polypeptides having Fc binding domains and two or more Fc-containing proteins, wherein each Fc-containing protein contains an Fc region; (b) purifying the multi-protein complex using a liquid chromatography method, wherein each Fc-containing protein in the purified multi-protein complex is bound to the Fc binding domain in the polypeptide; (c) contacting the cell with a composition comprising the purified multiprotein complex; The cells are T cells separated from peripheral blood mononuclear cells; wherein the two or more Fc-containing proteins are anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD3 antibodies and anti-CD28 antibodies; The molar ratio of the polypeptide to the two or more Fc-containing proteins is 1:3; The sequence of the polypeptide is shown in SEQ ID NO:

10.

2. A method for activating or expanding T cells, characterized in that: The method includes: (a) generating a multi-protein complex by mixing a polypeptide with two or more Fc-containing proteins, wherein the multi-protein complex contains two or more polypeptides having Fc binding domains and two or more Fc-containing proteins, wherein each Fc-containing protein contains an Fc region; (b) contacting the T cell with a composition comprising the multiprotein complex; wherein the two or more Fc-containing proteins are anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD3 antibodies and anti-CD28 antibodies, wherein the T cells are isolated from peripheral blood mononuclear cells, and wherein the molar ratio of the polypeptide to the two or more Fc-containing proteins is 1:3; The sequence of the polypeptide is shown in SEQ ID NO:

10.

3. The method according to claim 1, characterized in that The two or more Fc-containing proteins are antibodies that specifically bind to T cell surface antigens.

4. The method according to claim 1, wherein The two or more Fc-containing proteins provide a primary signal, a co-stimulatory signal, or a primary signal and a co-stimulatory signal for T cell activation.

5. The method according to claim 1, wherein wherein the two or more Fc-containing proteins are antibodies.

6. The method according to claim 1, characterized in that wherein the two or more Fc-containing proteins are heavy chain antibodies.

7. The method according to claim 1, characterized in that The two or more Fc-containing proteins are bispecific antibodies or Fc fusion proteins.

8. The method according to claim 1, characterized in that The multiprotein complex can bind to two or more target molecules in solution or on a solid surface.

9. The method according to claim 1, characterized in that The two or more Fc-containing proteins are antibodies that can specifically bind to one or more targets selected from CD3 and CD28.

10. The method according to claim 1 or 2, characterized in that The composition comprises two or more multi-protein complexes further assembled into a supercomplex through covalent bonds or non-covalent interactions.

11. The method according to claim 1 or 2, characterized in that The two or more Fc-containing proteins in the multiprotein complex are identical.

12. The method according to claim 1 or 2, characterized in that The composition comprises a plurality of multi-protein complexes, wherein the complexes are composed of two or more different Fc-containing proteins.

13. The method according to claim 1 or 2, characterized in that The composition comprises a plurality of multi-protein complexes, wherein each multi-protein complex comprises one or more anti-CD3 antibodies and one or more anti-CD28 antibodies.

14. The method according to claim 1 or 2, characterized in that wherein the T cells are CD8+ T cells.

15. The method according to claim 1 or 2, characterized in that in, The complex further comprises any one of anti-CD2 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD46 antibody, anti-CD137 antibody or a combination thereof.

16. A method for activating or expanding T cells, characterized in that The method comprises contacting a T cell with: a first multiprotein complex comprising a first polypeptide having two or more Fc binding domains, and two or more anti-CD3 antibodies, wherein the first multiprotein complex is generated by mixing a first polypeptide with the two or more anti-CD3 antibodies, wherein each anti-CD3 antibody binds to the Fc binding domain of the first polypeptide, and a second multiprotein complex comprising: a second polypeptide comprising two or more Fc binding domains and two or more anti-CD28 antibodies, wherein the second multi-protein complex is generated by mixing the second polypeptide with the two or more anti-CD28 antibodies, wherein each anti-CD28 antibody binds to an Fc binding domain in the second polypeptide; The T cells are isolated from peripheral blood mononuclear cells, wherein the molar ratio of the first polypeptide to the two or more anti-CD3 antibodies of the first multiprotein complex is 1:3, and the molar ratio of the second polypeptide to the two or more anti-CD28 antibodies of the second multiprotein complex is 1:3; the sequence of the polypeptide is shown in SEQ ID NO:

10.

17. The method according to claim 16, characterized in that The method further comprises: The T cell is contacted with a third multiprotein complex comprising a third polypeptide comprising two or more Fc-binding domains, and two or more Fc-containing proteins, each comprising an Fc region, wherein the third multiprotein complex is generated by mixing the third polypeptide with the two or more Fc-containing proteins, wherein each Fc-containing protein binds to an Fc-binding domain in the third polypeptide, and wherein the Fc-containing protein is an anti-CD28 antibody.

18. The method according to claim 16, characterized in that The two or more anti-CD3 antibodies in the first polypeptide complex are the same; and the two or more anti-CD28 antibodies in the second polypeptide complex are the same.

19. The method according to claim 16, wherein It further comprises contacting the T cells with an anti-CD2 antibody, an anti-CD7 antibody, an anti-CD27 antibody, an anti-CD46 antibody, an anti-CD137 antibody, an anti-CD226 antibody, or a combination thereof.

20. The method according to claim 16, wherein wherein the T cells are CD8+ T cells.

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