Functional binders synthesized and secreted by immune cells

Abstract

The present invention relates to an in vivo functional ligand (IFL) comprising a single-chain variable fragment (scFv) domain; a crystallizable fragment (Fc) domain; and a hinge domain connecting the scFv and Fc domains. The IFL specifically binds to a target receptor and can induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and cytokine stimulation. The IFL may be tethered to a chimeric antigen receptor via a self-cleaving peptide. The IFL may be expressed in immune cells such as natural killer cells or T lymphocytes. Vectors, host cells, and methods for producing the IFL are also described.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 875,455, filed Jul. 17, 2019. The entire teachings of the above application are incorporated herein by reference.

[0002] Incorporation by Reference of Data in ASCII Text File This application incorporates by reference the Sequence Listing contained in the following ASCII text file, filed concurrently herewith: a) File name: 44591154001_SEQUENCELISTING.txt; created on Jul. 15, 2020, size 35 KB.

Background Art

[0003] Cancer immunotherapy generally relates to inducing an immune response to selectively attack tumor cells. The toolbox of immunotherapies for treating cancer has been considerably enriched by the emergence of chimeric antigen receptor (CAR)-engineered T lymphocytes. Clinical experience with CAR - T cells demonstrates that T lymphocytes, when appropriately activated, can overcome resistance to chemotherapy, result in a significant reduction in tumor burden, disease stabilization, and in some patients with B - cell leukemia and lymphoma, can lead to tumor eradication. 18-26 In CAR - T cells, T - cell stimulation occurs via the expression of chimeric molecules with antibody - like properties.

[0004] Current CAR T - cell - based approaches do not fully utilize the potential of the immune system to target cancer cells.

Summary of the Invention

Means for Solving the Problems

[0005] Single-chain variable fragment (scFv) domain; crystallizable fragment (Fc) domain; and a peptide comprising a hinge domain connecting the scFv domain and the Fc domain are described herein. Nucleic acids encoding the peptides described herein; vectors comprising nucleic acids encoding the peptides described herein; immune cells (e.g., natural killer cells and T cells) expressing the peptides described herein; and methods for generating immune cells expressing the peptides described herein are also described.

[0006] The scFv domain can comprise an immunoglobulin variable light (V L ) domain, an immunoglobulin variable heavy (V H ) domain, and a linker domain connecting the V L and V H domains. The linker domain can be (G4S) x , where x is an integer from 1 to 100. The linker domain can be (G4S)3.

[0007] The scFv domain can bind to CD19, CD20, CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80, or CD86.

[0008] The Fc domain can comprise an immunoglobulin constant heavy 2 (C H 2) domain and an immunoglobulin constant heavy 3 (C H 3) domain. The Fc domain can be a human IgG1 Fc domain.

[0009] The peptide can further comprise a signal peptide at the N-terminus relative to the scFv domain.

[0010] The peptide can further include a self-cleaving peptide that connects the Fc domain to the chimeric receptor, and the chimeric receptor includes a receptor domain; a hinge and transmembrane domain; a co-stimulatory signaling domain; and a cytoplasmic signaling domain.

[0011] The self-cleaving peptide can be a 2A peptide. The receptor domain can be CD16. The hinge and transmembrane domain can be the CD8α hinge and transmembrane domain. The co-stimulatory domain can be the 4-1BB co-stimulatory domain. The cytoplasmic signaling domain can be CD3ζ cytoplasmic signaling. The chimeric receptor can be CD16V-4-1BB-CD3ζ.

[0012] In certain embodiments, the scFv domain binds to CD19 or CD20; the Fc domain is a human IgG1 Fc domain; and the hinge domain is an IgG1 hinge domain; the vector further includes a CD8α signal peptide that is N-terminal to the scFv domain; and the vector further includes a chimeric receptor that is CD16V-4-1BB-CD3ζ.

[0013] The vector can be a murine stem cell virus (MSCV).

[0014] The peptide can further include IL-15 linked to the Fc domain by a linker. The linker that links IL-15 to the Fc domain is SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK) z ;A(EAAAK) z A; and (XP) w selected from the group consisting of, z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100.

[0015] A peptide comprising a T cell receptor (TCR) β domain; a first crystallizable fragment (Fc) domain linked to the TCRβ domain; a TCRα domain; a self-cleaving peptide linking an Fc domain to the TCRα domain; and a second Fc domain linked to the TCRα domain is described herein. The peptide can further comprise a signal peptide linked to the T cell receptor (TCR) β domain. The first Fc domain can be the same as the second Fc domain. The first Fc domain can be different from the second Fc domain. Also described are nucleic acids encoding the peptide and vectors comprising the nucleic acids encoding the peptide.

[0016] Advantageously, the peptides described herein can be secreted by immune cells such as T cells and NK cells. As a result, immune cells can target and kill tumor cells without the need for in vitro administration of antibodies. When the secreted peptide binds to the Fc receptor on the surface of NK cells, the NK cells can exert antibody-dependent cell cytotoxicity. T cells transduced with the Fc receptor can also exert antibody-dependent cell cytotoxicity. Additionally, the peptide can cause phagocytosis of tumor cells by macrophages through interaction with the Fc receptor on the cell surface of macrophages. Finally, the peptide can kill tumor cells by inducing complement binding.

[0017] The foregoing will become apparent from the following more detailed description of the exemplary embodiments, as illustrated in the accompanying drawings in which like reference numerals refer to the same parts throughout the various figures. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

Brief Description of the Drawings

[0018]

FIG. 1A-C

FIG. 2

FIG. 3A-C

FIG. 4A-B

FIG. 5A-C

FIG. 6A-D

FIG. 7A-B

FIG. 8A-E

FIG. 9A-B

DETAILED DESCRIPTION OF THE INVENTION

[0019] The following describes the exemplary embodiments.

[0020] Monoclonal antibodies are essential for the latest cancer treatments. Antibodies exert anti-tumor activity through several mechanisms including direct induction of cell death, complement activation, and involvement of immune cells. Antibodies bound to tumor cells can cause antibody-dependent cell-mediated cytotoxicity (ADCC). 1-6 ADCC resulting from the involvement of Fc receptors (FcγR) expressed on the surface of natural killer (NK) cells 7 plays an important role in the clinical efficacy of antibodies; polymorphisms in the gene encoding FcγRIIIa (FCRG3A or CD16), which lead to receptors with higher affinity for Fc, have been associated with better anti-tumor effects in patients. 2,8-16 Other important mechanisms underlying the anti-tumor activity of antibodies include elimination of tumor cells by macrophages through antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). 7,17

[0021] The immunotherapy toolbox for treating cancer has been considerably enriched by the emergence of chimeric antigen receptor (CAR)-engineered T lymphocytes. Clinical experience with CAR-T cells demonstrates that T lymphocytes, when appropriately activated, can overcome resistance to chemotherapy and lead to significant reduction in tumor burden, disease stabilization, and in some patients with B-cell leukemia and lymphoma, tumor eradication. 18-26 In CAR-T cells, T cell stimulation occurs through the expression of a chimeric molecule with antibody-like properties. 27-31 Another approach leading to tumor-specific T cell activation is through the expression of high-affinity CD16 as a component of a chimeric receptor that includes stimulatory and costimulatory signals. 32 Such receptors have the potential to significantly enhance the antitumor effect of antibody therapy. Compared to CAR-T cells, it works in concert with other antibody-mediated mechanisms such as ADCP and CDC to produce a synergistic antitumor effect. Furthermore, by using multiple antibodies against a slightly expressed antigen, an active T cell response can be induced.

[0022] Methods are described herein that enable immune cells to produce binders with antibody-like functions. These in vivo functional ligands (IFLs) can cause ADCC, ADCP, and CDC as well as cytokine stimulation. These can be co-expressed with CD16 chimeric receptors in NK cells and T cells to optimize effector functions.

[0023] Certain embodiments described herein typically target CD20+ and CD19+ B cells, but this approach is applicable to targeting other antigens that are markers of cells contributing to the etiology of cancer and other diseases.

[0024] B-cell non-Hodgkin lymphoma and CD20 and CD19 B-cell non-Hodgkin lymphoma (NHL) is a cancer of lymphoid blood cells. NHL inevitably progresses and is fatal if untreated. Standard treatments include chemotherapy, antibody therapy, therapy with tyrosine kinase inhibitors, and hematopoietic stem cell transplantation. CD20 and CD19 are B-cell specific antigens that are widely expressed in B-cell NHL (also referred to as B-NHL).

[0025] The vectors described herein can be used to produce modified T cells, which can further be used for the targeted treatment of NHL. The processes described herein can be targeted to destroy CD20+ and CD19+ B cells, thereby creating transgenic T cells that can eradicate NHL and / or reduce its severity.

[0026] Acute lymphoblastic leukemia and CD19 Acute lymphoblastic leukemia (ALL) is also a cancer of lymphoid blood cells. ALL progresses rapidly and is fatal if untreated. Standard treatments include chemotherapy and hematopoietic stem cell transplantation. CD19 is a B-cell specific antigen that is expressed on all leukemia cells in the majority of cases of ALL.

[0027] The vectors described herein can be used to produce modified T cells, which can further be used for the targeted treatment of ALL. The processes described herein can be targeted to destroy CD19+ B cells, thereby creating transgenic T cells that can eradicate ALL and / or reduce its severity.

[0028] Nucleic acid As used herein, the term "nucleic acid" refers to a polymer comprising a number of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acids" include, for example, DNA (e.g., genomic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, "nucleic acid" can refer to one or both strands of the molecule.

[0029] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers and non-naturally occurring derivatives and analogs thereof. Thus, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0030] As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have the same residue at the same position when the sequences are aligned to achieve the maximum level of identity expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence and compared to a test sequence. The sequence identity between the reference sequence and the test sequence is expressed as a percentage of the positions over the full length of the reference sequence that share the same nucleotide or amino acid when the reference sequence and the test sequence are aligned to achieve the maximum level of identity. As an example, when aligned to achieve the maximum level of identity, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the full length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0031] The alignment of arrays for comparison to achieve maximum level identity can be readily performed by one of ordinary skill in the art using an appropriate alignment method or algorithm. In some cases, the alignment can include introduced gaps in order to yield the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).

[0032] When using an array comparison algorithm, input the test array and the reference array into the computer, and if necessary, specify subsequent coordinates and the array algorithm program parameters. Then, calculate the percent sequence identity for the test array compared to the reference array based on the specified program parameters by the array comparison algorithm. A commonly used tool for determining the percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP) available from the National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., J Mol Biol. 215(3):403-10(1990)).

[0033] In various embodiments, two nucleotide sequences or two amino acid sequences can have at least, for example, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity. When ascertaining the percent sequence identity to one or more sequences described herein, the sequences described herein serve as the reference sequences.

[0034] Vector The terms "vector", "vector construct", and "expression vector" mean a means of transporting a DNA sequence or RNA sequence (e.g., a foreign gene) into a host cell so as to transform the host and facilitate the expression (e.g., transcription and translation) of the introduced sequence. Vectors typically contain DNA, a transmissible material, into which exogenous DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is the "plasmid", which is generally an independent molecule of double-stranded DNA that can readily accept additional (exogenous) DNA and can be easily introduced into a suitable host cell. Many vectors, including plasmids and fungal vectors, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts.

[0035] The terms "express" and "expression" mean to enable or cause the information in a gene and DNA sequence to appear, for example, to produce a protein by activating the functions of a cell involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or near a cell to form an "expression product" such as a protein. The expression product itself, such as the resulting protein, can also be said to be "expressed" by the cell. A polynucleotide or polypeptide is recombinantly expressed, for example, if it is expressed or produced in a foreign host cell under the control of a foreign or native promoter or in a native host cell under the control of a foreign promoter. A gene delivery vector generally contains a transgene (e.g., a nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for the expression of the transgene in the host cell into which the vector is introduced. Promoters and delivery constructs suitable for gene expression are known in the art. Recombinant plasmids can also contain an inducible or regulatable promoter for the expression of an enzyme in a cell.

[0036] A variety of gene delivery vehicles are known in the art and include viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, for example, vectors derived from herpes virus, baculovirus vectors, lentivirus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors (AAV), and murine stem cell virus (MSCV). The viral vector can be a replicating or non-replicating viral vector. Such vectors can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.

[0037] Non-viral vectors for gene delivery include, inter alia, naked DNA, plasmids, transposons, and mRNA. Non-limiting examples include the pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.), pMAL plasmid (New England Biolabs, Beverly, Mass.). Such vectors can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.

[0038] In certain embodiments, the vector includes an internal ribosome entry site (IRES) within the sequence. In some embodiments, the vector includes a selectable marker such as the ampicillin resistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein such as green fluorescent protein (GFP) or mCherry. In some embodiments, the nucleic acid is suitable for subcloning into pMSCV-IRES-GFP between EcoRI and XhoI. In some embodiments, the vector contains a multiple cloning site (MCS) for insertion of the desired gene.

[0039] The genetic code is degenerate in that most amino acids are represented by multiple codons (referred to as "synonymous" or "same sense" codons), but it is understood in the art that the codon usage frequency by a particular organism is non-random and biased towards certain triplet codons. Thus, in some embodiments, the vector contains a nucleotide sequence optimized (e.g., through codon optimization) for expression in a particular type of host cell. Codon optimization means the process by which a polynucleotide encoding a protein of interest is modified such that certain codons in the polynucleotide are replaced with codons that encode the same amino acid but are commonly used / recognized in the host cell in which the nucleic acid is being expressed. In some aspects, the polynucleotides described herein are codon optimized for expression in T cells.

[0040] In vivo functional ligand Figure 1A is a schematic diagram of an in vivo functional ligand (IFL) construct. The IFL includes a single-chain variable fragment (scFv) domain, a modified crystallizable fragment (Fc) domain, and a hinge domain connecting the scFv and the modified Fc domain. Preferably, an N-terminal signal peptide (leader peptide) that directs the IFL towards the secretory pathway and is ultimately secreted by the cell is included. Signal peptides of surface proteins are generally suitable, and by way of example, there is the CD8α signal peptide.

[0041] The scFv domain typically includes an immunoglobulin variable light (V L ) domain, an immunoglobulin variable heavy (V H ) domain, and a linker domain connecting the V L and V H domains. The relative positions of the V L and V H domains can be reversed, but they are both N' to the modified Fc domain as shown in Figure 1A.

[0042] The scFv domain targets antigens of interest, such as antigens on tumor cells. Certain scFvs described herein are anti-CD19 single-chain variable fragments (anti-CD19 scFv). Another certain scFv described herein is an anti-CD20 single-chain variable fragment (anti-CD20 scFv).

[0043] The embodiments described herein relate to anti-CD19 constructs and anti-CD20 constructs, but a similar approach can be applied to produce constructs against other target antigens such as CD22, CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD)-2, PD-L1 (CD274), CD80, or CD86. For example, based on the schematic diagram in FIG. 5A, the anti-CD19 scFv portion can be exchanged with various scFvs that specifically bind to various target antigens. Other targets including CD22, CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80, or CD86 are suitable.

[0044] The hinge domain connects the scFv and the modified Fc domain, and in some cases, the hinge domain may be considered part of the Fc domain. An example of the hinge domain is the IgG hinge domain. The construct can also include an N-terminal signal peptide such as the CD8α signal peptide (see SEQ ID NOs: 21 and 22).

[0045] V L and V H Various linker domains between the domains are suitable. In some embodiments, the linker domain is (G4S) xIt can be set that x is an integer from 1 to 100; preferably, x is an integer from 1 to 10; even more preferably, x is an integer from 2 to 5. In some embodiments, the linker domain can be (G4S)3. In other embodiments, the linker domain can be one or more glycine residues (e.g., (G) y It can be set that y is an integer from 2 to 100. In other embodiments, the linker domain is (EAAAK)3, (G4S) x , (G4S)3, and (G) y It can be set, and as an example of a flexible linker, (EAAAK)3 is an example of a more rigid linker.

[0046] Various hinge domains are suitable. In some embodiments, the hinge domain can be an IgG hinge domain. In some embodiments, the hinge can be a plurality of amino acid residues. In some embodiments, the hinge domain can be a hinge domain derived from IgE, IgA, IgD, or CD8α.

[0047] In some embodiments, the construct is a bicistronic vector that also encodes a chimeric receptor, as shown in Figure 6A. The chimeric receptor can include a receptor domain, a hinge and transmembrane domain, a co-stimulatory signaling domain, and a cytoplasmic signaling domain. In the construct of Figure 6A, the chimeric receptor is linked to a modified Fc domain by a 2A peptide, which is a self-cleaving peptide. By linking the scFv domain and the Fc domain to the chimeric receptor, the co-expression of both proteins can be achieved from a single vector. Examples of 2A peptides are P2A (SEQ ID NOs: 43 and 44), T2A (SEQ ID NOs: 45 and 46), E2A (SEQ ID NOs: 47 and 48), and F2A (SEQ ID NOs: 49 and 50), but other 2A peptides are known in the art.

[0048] Modifications to the design of IFL The design of the IFL construct being tested in this study can be further modified to enhance some of its functions and / or broaden the range of its specificity. For example, the modified Fc can be further engineered to increase its affinity for Fc receptors in NK cells and macrophages, and thus enhance ADCC and ADCP, and / or to increase its ability to bind complement. 41-43

[0049] In one modification (Figure 8A), examples of two mutations introduced into the C H 2 domain of Fc (serine instead of aspartic acid at position 239, S239D; and isoleucine instead of glutamic acid at position 332, I332E) are made to improve affinity for Fc receptors; two other mutations (S267E and H268F) are made to increase complement binding. 41,43

[0050] In another modification (Figure 8B), examples of mutations introduced into the C H 2 domain of Fc (E345K, E430G, and S440Y) are made to promote hexamer formation. The IFL hexamer may be able to increase ADCC and CDC. 44,48

[0051] In another modification (Figure 8C), IL-15 is added to the IFL construct; this cytokine promotes activation and expansion of immune cells. 45,46 IFL and IL-15 are linked by a linker. Various linker domains between the IFL construct and the cytokine are suitable. In some embodiments, the linker domain is the amino acid of SEQ ID NO: 52 produced by its corresponding nucleotide sequence (SEQ ID NO: 51). In some embodiments, the linker domain can be A(EAAK)4ALEA(EAAAK)4A. In other embodiments, the linker domain is (EAAAK) z and A(EAAAK) zLet it be A, and z is an integer from 1 to 100; preferably, z is an integer from 2 to 5. In other embodiments, the linker domain is (XP) w Let it be, where X specifies any amino acid; preferably, X is alanine, lysine, or glutamic acid, and w is an integer from 1 to 100.

[0052] In another modification (FIG. 8E), a ligand that binds to a co-stimulatory molecule of immune cells such as 4-1BB (CD137), CD28, or OX40 (CD134) is added to the IFL construct. The IFL and the co-stimulatory ligand are linked by a linker. Various linker domains between the IFL construct and the co-stimulatory ligand are suitable and are generally the same as the linker domains suitable between the IFL construct and the cytokine in FIG. 8C.

[0053] FIG. 8D shows a construct in which the binding domain of IFL is the extracellular domain of a T cell receptor (TCR) induced against Epstein - Barr virus. 47 Such an IFL can recognize peptides produced by virus - infected or oncogenically transformed cells and can be expressed on the cell membrane in association with MHC / HLA molecules. Therefore, such an IFL could be used to target viral peptides or peptides produced by cancer cells that cannot be recognized by antibodies or scFvs derived from antibodies.

[0054] Method for producing transgenic host cells Methods for producing transgenic host cells such as transgenic natural killer (NK) cells or transgenic T cells are described herein. Transgenic host cells can be produced, for example, by introducing one or more vector embodiments described herein into a host cell.

[0055] In one embodiment, the method includes introducing into a host cell a vector comprising a nucleic acid encoding an IFL. In some embodiments, the nucleic acid, such as a bicistronic vector, expresses the IFL together with a chimeric receptor. In some embodiments, two separate vectors can be used to create transgenic cells, such as transgenic T cells, that express the IFL and the chimeric receptor.

[0056] In some embodiments, one or more nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acid integrated into the host genome can be introduced into the host cell using any of a variety of suitable techniques known in the art, such as homologous recombination, CRISPR-based systems (e.g., CRISPR / Cas9; CRISPR / Cpf1), and TALEN systems.

[0057] Host cell A variety of host cells are suitable for generating transgenic host cells. Most commonly, the host cell is an immune cell, such as a natural killer (NK) cell or a T lymphocyte cell.

[0058] As used herein, "natural killer cell" ("NK cell") refers to a type of cytotoxic lymphocyte of the immune system. NK cells effect a rapid response against virus-infected cells and respond to cancerous cells. Typically, immune cells detect peptides derived from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering cytokine release and causing lysis or apoptosis. However, NK cells are unique in that they have the ability to recognize stressed cells regardless of whether peptides derived from pathogens are present on MHC molecules. NK cells were named "natural killer" because they were initially thought not to require prior activation to kill their targets. NK cells are large granular lymphocytes (LGLs) known to differentiate and mature in the bone marrow, and then enter the bloodstream from the bone marrow. When antibodies are bound to antigens on the surface of tumor cells, NK cells can also kill the tumor cells; the Fc portion of the antibody binds to Fc receptors (CD16) on the surface of NK cells, triggering a cytotoxic effect in a process known as antibody-dependent cell cytotoxicity (ADCC).

[0059] As used herein, "T lymphocyte" or "T cell" refers to lymphocytes that mature in the thymus. T cells can be further characterized and divided into subpopulations including T helper (T H ) cells, cytotoxic T (T C ) cells, and regulatory T (T reg ) cells. T H and T C cells can be characterized according to the presence or absence of the membrane glycoproteins CD4 and CD8. Generally, T H cells express CD4 on their surface, while T C cells express CD8 on their surface. T helper cells can be further characterized as T H 1 cells and T H 2 cells. T cells can also exhibit ADCC when transduced with CD16 and receptors encoding signaling molecules.32

[0060] In some embodiments, the NK cells or T cells are mammalian cells. Examples of "mammal" or "mammalian" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, and other like species. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In certain embodiments, the mammalian T or NK cells are human T or NK cells.

[0061] When a vector containing a nucleic acid encoding IFL is introduced into a host cell, the host cell becomes a transgenic host cell that expresses IFL. Typically, IFL is secreted by the transgenic host cell.

[0062] Values and Ranges Unless otherwise specified or unless otherwise clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can be considered to be any specific value or sub-range within the ranges specified in various embodiments, unless the context clearly dictates otherwise. "About" with respect to a numerical value generally refers to a range of values within ±8% of that value, in some embodiments ±6%, in some embodiments ±4%, in some embodiments ±2%, in some embodiments ±1%, in some embodiments ±0.5% of that value, unless otherwise specified or unless otherwise clear from the context.

Examples

[0063] Materials and Methods Cells The human cell lines RS4;11 and Nalm-6 (B-cell leukemia), Ramos, Raji, and Daudi (B-cell lymphoma), and Jurkat (T-cell leukemia) were obtained from the American Type Culture Collection (Rockville, MD). The B-cell leukemia cell line OP-1 was established in the inventors' laboratory. 33The inventors transduced Nalm-6 and Daudi with a murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retroviral vector (from Vector Development and Production Shared Resource of St. Jude Children’s Research Hospital, Memphis, TN) containing the firefly luciferase gene. The inventors also transduced Ramos and Raji with an MSCV retroviral vector containing the mCherry gene. The transduced cells were sorted for their GFP or mCherry expression, respectively, using a MoFlo cell sorter (Beckman Coulter, Brea, CA). To make Nalm-6 express CD20 on the surface, the inventors subcloned the human CD20 gene in a cytomegalovirus plasmid (pCMV6) vector (Origene, Rockville, MD) into the MSCV-IRES-GFP vector and transduced Nalm-6 with the CD20 gene. Nalm-6 cells expressing CD20 were selected using a MoFlo cell sorter after staining with an anti-CD20 antibody (BD Biosciences, San Jose, CA). The cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin.

[0064] Peripheral blood was obtained from the waste of donated platelets from healthy donors at the National University Hospital Blood Bank, Singapore. Mononuclear cells were isolated by density gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway) and washed twice in RPMI-1640. For viral transduction, NK cells were expanded from the isolated mononuclear cells with a genetically modified K562-mb15-41BBL previously established in the inventors' laboratory. 34,35T cells were activated by T cell TransAct (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in TexMACS medium (Miltenyi Biotec) containing interleukin-2 (IL-2, Proleukin, Novartis, Basel, Switzerland, 100 IU / mL).

[0065] Plasmid and viral transduction The inventors designed an IFL composed of a single-chain variable fragment (scFv) linked to a modified crystallizable fragment domain (Fc) of human immunoglobulin G1 (IgG1). The amino acid sequences of the signal peptide, scFv against CD20, and modified Fc of IgG1 were obtained from the sequence of rituximab described in DrugBank (http: / / www.drugbank.ca; accession number DB00073). The scFv sequence against CD19 was derived from the anti-CD19-41BB-CD3ζ CAR previously developed in the inventors' laboratory. 31 The variable domains of the heavy and light chains were joined by a flexible linker sequence encoding (Gly4Ser)3. The linked scFv was connected to the signal peptide and hinge, followed by the constant heavy domains 2 and 3 (C H 2, C H 3) of IgG1. The anti-CD20 IFL was fused with CD16V-4-1BB-CD3ζ, which could induce ADCC in T cells through the self-cleaving 2A peptide (P2A) as previously described by the inventors' laboratory. 36 The gene was subcloned into the MSCV vector with or without GFP.

[0066] Gene transduction by the retroviral vector was performed as previously described. 37Briefly, the MSCV retroviral vector was added to RetroNectin-coated (Takara, Otsu, Japan) tubes and incubated at 4°C for 16 hours. Activated NK cells or T lymphocytes were then added to the tubes after removal of the supernatant and incubated at 37°C in 5% CO2 for 24 hours. The transduction procedure was repeated once more the next day. The transduced cells were maintained in RPMI-1640 with 10% FB and IL-2.

[0067] Determination of IFL expression and specificity To detect IFL expression, transduced cells were permeabilized with 8E reagent (a permeabilization reagent developed in the inventors' laboratory) and then stained with phycoerythrin (PE)-conjugated anti-human IgG antibody (SouthernBiotech, West Grove, PA). Cell surface CD16 and CD3 expression was determined by anti-CD16-PE (clone B73.1, BD Biosciences) and anti-CD3-APC (clone SK7, BD Biosciences), respectively.

[0068] For the specificity of IFL, culture supernatants from transduced cells were added to Jurkat (CD20 negative, CD19 negative), Ramos (CD20 positive, CD19 positive), or RS4;11 (CD20 negative, CD19 positive) at 1 μg / mL and incubated for 10 minutes. IFL bound to the cell surface was detected with PE-conjugated anti-human IgG antibody. Cell staining was analyzed using a BD LSRFortessa (BD Biosciences).

[0069] Measurement of IFL concentration and glycosylation analysis The IFL concentration in the culture supernatant derived from the transfected cells was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the culture supernatant containing IFL or rituximab was incubated on plates coated with a PE-conjugated anti-human IgG antibody for 1 hour and then washed. Subsequently, a horseradish peroxidase (HRP)-conjugated anti-rituximab antibody (MB2A4, Bio-rad, Hercules, CA) was added to the plates and incubated for 1 hour. Fluorescence was measured by Infinite 200 PRO (Tecan, Mannedorf, Switzerland) after adding QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher). The IFL concentration was determined by a calibration curve prepared with rituximab.

[0070] Glycosylation analysis was performed by Proteodynamics (Riom, France). Briefly, IFL in the culture supernatant of the transfected cells was concentrated by a dialysis membrane (Amicon Ultra-15 Centrifugal Filter Units, Merck Millipore, Burlington, MA) and purified using an NAB Protein G Spin kit (Thermo Fisher). The purified IFL was denatured in 0.5% sodium dodecyl sulfate (SDS) and 1% β-mercaptoethanol and deglycosylated with PNGase F (Promega, Fitchburg, WI). The N-glycans released by PNGase were purified by Hypercarb Hypersep 200mg (Thermo Fisher) and permethylated with sodium hydroxide, dimethyl sulfoxide (DMSO), and methyl iodide (ICH3) prior to MALDI-TOF MS analysis using an Autoflex speed mass spectrometer (Bruker, Billerica, MA).

[0071] Cytotoxicity assay in vitro For the CDC assay, Ramos or SUDHL-4 in RPMI / 10% FBS medium with or without 5% complement (Sigma-Aldrich, Saint Louis, MO) was plated, and rituximab or anti-CD20 IFL was added at 0.05 μg / ml. Live cells were counted by Accuri CD6 (BD Biosciences) after 2-hour incubation at 37 °C in 5% CO2.

[0072] To test ADCP, Ramos cells labeled with mCherry were cultured with or without THP-1 at a 1:1 ratio for 48 hours in the presence of 0.1 μg / ml anti-CD20 IFL or rituximab. Ramos cells were counted by the IncuCyte Zoom System (Essen BioScience, Ann Arbor, MI).

[0073] For the ADCC assay, target cells stained with calcein AM (Thermo Fisher) were co-cultured with transduced NK cells or T lymphocytes at a 2:1 effector-to-target (E:T) ratio for 4 hours. Target live cells were counted by flow cytometry. In other experiments, target cells expressing mCherry were incubated with NK cells or T lymphocytes with IL-2 (200 IU / mL for NK cells and 100 IU / mL for T cells) at 37 °C in 5% CO2. As a control, rituximab was added at 1.0 μg / ml to NK cells having only GFP. Target cells were counted every 8 hours for 3 days using the IncuCyte Zoom System.

[0074] IFL Kinetics and Dynamics in Mouse Models To measure the plasma concentration of IFL secreted from T cells, NOD.Cg-Prkdc scid IL2rg tm1Wjl2×10 T cells transduced with anti-CD20 IFL-P2A-CD16V-4-1BB-CD3ζ were injected into / SzJ (NOD / scid IL2RGnull) mice (The Jackson Laboratory, Bar Harbor, ME). 7 Subsequently, 2 days later, 2×10 5 Nalm-6 cells expressing CD20 were injected intravenously (i.v.). The mice also received 20,000 IU of IL-2 intraperitoneally every 2 days for 3 weeks. IFL in plasma was measured by ELISA.

[0075] To test for antitumor activity in vivo, luciferase-labeled Daudi cells were injected intraperitoneally (i.p.) at 2×10 5 cells per mouse into NOD / scid IL2RGnull mice. 3 and 6 days later, the mice received T cells transduced with anti-CD20 IFL-P2A-CD16V-4-1BB-CD3ζ at 2×10 7 cells per mouse i.p. Other mice received 2×10 7 T cells transduced with GFP or 0.2 ml of RPMI 1640 only instead of T cells. All mice received 20,000 IU of IL-2 every 2 days for 1 or 3 weeks. Growth of Daudi cells was measured using the Xenogen IVIS-200 System (Caliper Life Sciences, Waltham, MA) after injection of D-luciferin potassium salt (Perkin Elmer, Waltham, MA). Luminescence was analyzed with Living Image 3.0 software (Perkin Elmer). Mice were euthanized when luminescence reached 1×10 11 photons per second or when physical signs justifying euthanasia appeared.

[0076] Results Design and expression of IFL The inventors first produced an scFv fragment from the sequence published for the anti-CD20 antibody rituximab. This scFv and an anti-CD19 scFv previously developed in the inventors' laboratory31 was linked to the hinge and heavy chain constant domains 2 (C H 2) and 3 of human IgG1 (C H 3) (Figure 1A). The inventors inserted the IFL gene into an MSCV retroviral vector that also contained the GFP gene and transduced them into expanded NK cells. To determine whether IFL was synthesized by the transduced cells, the inventors performed intracellular staining targeting the Fc component. As shown in Figure 1B, most NK GFP+ cells also expressed anti-CD20 IFL or anti-CD19 IFL. Similar results were seen when the same constructs were transduced into peripheral blood T lymphocytes (Figure 1C).

[0077] The inventors determined whether IFL could bind to its cognate target. As shown in Figure 2, secreted anti-CD19 IFL labeled CD19+ cells Ramos and RS4;11, while anti-CD20 IFL labeled only the CD20+ Ramos cell line and did not label the CD20− RS4;11 cell line. Neither labeled the CD19− CD20− T cell line Jurkat (Figure 2).

[0078] Characterization of IFL To measure the ability of immune cells to produce IFL, the inventors collected culture medium from cells transduced with anti-CD20 IFL and measured the antibody concentration by ELISA using an anti-idiotype rituximab antibody. As shown in Figure 3A, both NK cells and T cells secreted anti-CD20 IFL. In particular, the amount of IFL measured in the supernatant of T cells was significantly higher than the amount measured in the supernatant of NK cells (P < 0.01). The amount of IFL secreted from 1 × 10 6 NK cells in 24 hours was equivalent to 23.5 ng of rituximab (range, 15.1 - 36.8 ng, n = 3). The amount of IFL secreted by T cells was equivalent to 74.3 ng (range, 62.8 - 93.2 ng, n = 3).

[0079] To clarify the type of post-translational modification profile of constructs produced by immune cells, the inventors performed an analysis of N-linked glycans bound to the modified Fc domain using MALDI-TOF. Twelve N-glycan structures were detected for NK cell IFL, and eight were detected for T cell IFL. In both cases, the major structure was a disialylated biantennary N-glycan that has no core fucose and contains two galactoses and two terminal sialic acids, named G2S2 ([M+Na] + 2792). Interestingly, 79% and 59% of the Fc glycans were non-fucosylated when IFL was produced by NK and T cells, respectively. (Figure 3B, C). As a control, the inventors also tested the N-linked glycan pattern of rituximab; the N-glycans detected were two fucosylated biantennary N-glycans ([M+Na]+1836 = G0F, 2040 = G1F).

[0080] IFL mediates CDC, ADCP, and ADCC To test whether IFL can mediate CDC, the inventors incubated CD20+ B-lymphoma cell lines Ramos, SUDHL-4, and Raji with various concentrations of anti-CD20 IFL (collected from the supernatants of NK or T cells transduced with IFL) and 5% complement for 2 hours. In a parallel test, anti-CD20 IFL was replaced with rituximab. As shown in Figure 4A, IFL caused substantial lysis of both the Ramos and SUDHL-4 cell lines (which are known to be sensitive to complement lysis), while the complement-resistant Raji cells remained largely unaffected. 38,39

[0081] ADCP was tested by co-culturing the monocytic cell line THP-1, which can exert phagocytosis of tagged target cells, with Ramos. 40 As shown in Figure 4B, IFL derived from NK or T cells was able to promote the disappearance of Ramos cells in the presence of THP-1 cells.

[0082] To determine whether IFL produced by NK cells and T cells can mediate ADCC, the inventors co-cultured NK cells transduced with either GFP alone or anti-CD20 IFL with the CD20+ lymphoma cell line Raji at an E:T ratio of 1:1, using NK-GFP cells and 1 μg / mL rituximab as controls. As shown in Figure 5A, the IFL NK cells exerted a potent cytotoxic effect. In other tests, the inventors determined the cytotoxic ability of NK cells transduced with anti-CD19 IFL against three CD19+ leukemia cell lines (RS4;11, OP-1, and Nalm-6). As shown in Figure 5B, the NK-IFL cells were significantly more potent than NK cells transduced with GFP alone, and cell killing against the CD19+CD20- cell line RS4;11 was mediated only by anti-CD19 IFL (Figure 5C).

[0083] T cells expressing the CD16 receptor exert ADCC through self-produced IFL The inventors prepared a bicistronic construct containing anti-CD20 IFL separated by P2A and the CD16(V158)-41BB-CD3ζ receptor (Figure 6A). CD16-41BB-CD3ζ was previously produced in the inventors' laboratory and has been shown to confer the ability of ADCC to T lymphocytes. 32 The inventors transduced T lymphocytes with a construct that achieves the expression of both components (Figure 6B, C). When exposed to Ramos cells in long-term culture, T lymphocytes expressing both IFL and CD16-41BB-CD3ζ eradicated the lymphoma cells, while T cells expressing only one of the genes or GFP did not (Figure 6D). Additional information regarding CD16-41BB-CD3ζ can be found in U.S. Patent No. 10,144,770 B2 and U.S. Patent Application Publication No. 2015 / 0139943, both of which are hereby incorporated by reference in their entirety.

[0084] Next, the inventors determined the levels of plasma IFL that can be measured in mouse plasma after intravenous injection of 2×10 7 T lymphocytes transduced with anti-CD20 IFL. As shown in FIG. 7A, IFL could be detected in plasma 50 days after cell injection, indicating that IFL secretion is persistent. The inventors evaluated whether T lymphocytes expressing both IFL and CD16-41BB-CD3ζ could exert antitumor activity in NOD-SCID-IL2RGnull immunodeficient mice transplanted intraperitoneally with the CD20+ B cell lymphoma cell line Daudi. After intraperitoneal injection of T cells, there was strong antitumor activity in mice that received T cells with IFL and CD16-41BB-CD3ζ, but the tumors grew rapidly in mice that received T cells transduced with GFP only or no T cells (FIG. 7B).

[0085] Modified IFL The IFL construct was modified to enhance some of its functions and / or broaden the range of its specificity. For example, the modified Fc can be further modified to increase its affinity for Fc receptors in NK cells and macrophages, and thus to enhance ADCC and ADCP and / or to increase its ability to bind complement. In particular, the modified IFLs of FIGS. 8A-D were constructed.

[0086] The results of the experiments shown in FIGS. 9A - B demonstrate that adding a sequence encoding IL - 15 to the anti - CD20 IFL secreted by NK cells significantly increases the killing activity against CD20+ lymphoma cells in a 3 - day co - culture. In these experiments, when NK cells were transduced with IFL - IL15, the number of Ramos cells decreased most effectively; these cells were more potent than cells transduced with ILF lacking IL - 15, and they were further more potent than NK cells transduced with GFP only. The superiority of IFL - IL15 was observed regardless of whether IL - 2 was present in the culture. These results demonstrate that the function of IFL can be enhanced by linking it to other functional molecules.

[0087] Additional Embodiments 1. A peptide comprising the following: a) A single - chain variable fragment (scFv) domain; b) A crystallizable fragment (Fc) domain; and c) A hinge domain connecting the scFv domain and the Fc domain.

[0088] 2. The peptide of Embodiment 1, wherein the scFv domain comprises an immunoglobulin variable light (V L ) domain, an immunoglobulin variable heavy (V H ) domain, and a linker domain connecting the V L and V H domains.

[0089] 3. The peptide of Embodiment 2, wherein the linker domain is (G4) x where x is an integer from 1 to 100.

[0090] 4. The peptide of Embodiment 3, wherein the linker domain is (G4)3.

[0091] 5. The peptide of any one of Embodiments 1 - 4, wherein the scFv domain binds to CD19.

[0092] 6. The scFv domain is a peptide according to any one of Embodiments 1 to 4 that binds to CD20.

[0093] 7. The scFv domain is a peptide according to any one of Embodiments 1 to 4 that binds to CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80, or CD86.

[0094] 8. The Fc domain is a peptide according to any one of Embodiments 1 to 4 that comprises an immunoglobulin constant heavy 2 (C H 2) domain and an immunoglobulin constant heavy 3 (C H 3) domain.

[0095] 9. The Fc domain is a peptide according to any one of Embodiments 1 to 4 that is a human IgG1 Fc domain.

[0096] 10. A peptide according to any one of Embodiments 1 to 4 that further comprises a signal peptide at the N-terminus with respect to the scFv domain.

[0097] 11. A peptide according to any one of Embodiments 1 to 4 that further comprises a self-cleaving peptide that connects the Fc domain to the chimeric receptor, and the chimeric receptor comprises a receptor domain, a hinge and transmembrane domain, a co-stimulatory signaling domain, and a cytoplasmic signaling domain.

[0098] 12. The self-cleaving peptide is a 2A peptide, which is the peptide of Embodiment 11.

[0099] 13. The receptor domain is CD16, which is the peptide of Embodiment 11.

[0100] 14. The hinge and transmembrane domain is the CD8α hinge and transmembrane domain, which is the peptide of Embodiment 11.

[0101] 15. The co-stimulatory domain is the 4-1BB co-stimulatory domain, and the peptide of embodiment 11.

[0102] 16. The cytoplasmic signaling domain is the CD3ζ cytoplasmic signaling, and the peptide of embodiment 11.

[0103] 17. The chimeric receptor is CD16V-4-1BB-CD3ζ, and the peptide of embodiment 11.

[0104] 18. The scFv domain binds to CD19 or CD20, the Fc domain is the human IgG1 Fc domain, and the hinge domain is the IgG1 hinge domain; the peptide further comprises a CD8α signal peptide at the N-terminus with respect to the scFv domain; the peptide further comprises a chimeric receptor which is CD16V-4-1BB-CD3ζ, and the peptide of any one of embodiments 1 to 4.

[0105] 19. The peptide of any one of embodiments 1 to 4 further comprises one or more of the following mutations: S239D; S267E; H268F; or I332E.

[0106] 20. The peptide of any one of embodiments 1 to 4 further comprises one or more of the following mutations: E345K; E430G; or S440Y.

[0107] 21. The peptide of any one of embodiments 1 to 4 further comprises IL-15 linked to the Fc domain by a linker.

[0108] 22. The linker connecting IL-15 to the Fc domain is SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK) z ; A(EAAAK) z A; and (XP) w selected from the group consisting of, z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100, and the peptide of embodiment 21.

[0109] 23. The peptide is the peptide of any one of Embodiments 1 to 4, further comprising a ligand that binds to 4-1BB (CD137), CD28, or OX40 (CD134) and is linked to the Fc domain by a linker.

[0110] 24. The linker that connects IL-15 to the Fc domain is SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK) z ; A(EAAAK) z A; and (XP) w selected from the group consisting of, z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100, the peptide of Embodiment 23.

[0111] 25. A nucleic acid encoding the peptide of any one of Embodiments 1 to 24.

[0112] 26. A vector comprising the nucleic acid encoding the peptide of any one of Embodiments 1 to 24.

[0113] 27. The vector of Embodiment 26, wherein the vector is murine stem cell virus (MSCV).

[0114] 28. An immune cell expressing the peptide, wherein the peptide a) T cell receptor (TCR) β domain; b) a first crystallizable fragment (Fc) domain linked to the TCRβ domain; c) TCRα domain; d) a self-cleaving peptide that connects the Fc domain to the TCRα domain; e) a second Fc domain linked to the TCRα domain comprises, the immune cell.

[0115] 29. The immune cell of Embodiment 28, further comprising a signal peptide linked to the T cell receptor (TCR) β domain.

[0116] 30. The immune cell of embodiment 28, wherein the first Fc domain is the same as the second Fc domain.

[0117] 31. A peptide comprising: a) A T cell receptor (TCR) β domain; b) A first crystallizable fragment (Fc) domain linked to the TCRβ domain; c) A TCRα domain; d) A self-cleaving peptide that links an Fc domain to the TCRα domain; e) A second Fc domain linked to the TCRα domain.

[0118] 32. The peptide of embodiment 31, further comprising a signal peptide linked to the T cell receptor (TCR) β domain.

[0119] 33. The peptide of embodiment 31, wherein the first Fc domain is the same as the second Fc domain.

[0120] 34. A nucleic acid encoding the peptide of any one of embodiments 31-33.

[0121] 35. A vector comprising a nucleic acid encoding the peptide of any one of embodiments 31-33.

[0122] 36. A method for producing a transgenic host cell, comprising introducing a vector into the host cell, the vector comprising a nucleic acid encoding the peptide of any of embodiments 1-24 or embodiments 31-33.

[0123] 37. A method for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) in a subject, comprising administering to the subject a therapeutically effective amount of any of the immune cells described herein.

[0124] Sequence Accession No. 1: Anti-CD20 IFL, rituximab signal peptide; cDNA: ATGGACTTCCAGGTGCAGATCATCAGCTTTCTGCTGATCTCCGCCTCT

[0125] Accession No. 2: Anti-CD20 IFL, rituximab signal peptide; Amino acid: MDFQVQIISFLLISAS

[0126] Accession No. 3: Anti-CD20 IFL, immunoglobulin variable domain of rituximab light chain; cDNA:

Chem.

[0127] Accession No. 4: Anti-CD20 IFL, immunoglobulin variable domain of rituximab light chain; Amino acid: VIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0128] Accession No. 5: Anti-CD20 IFL, linker; cDNA: GGCGGCGGCGGCTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCC

[0129] Accession No. 6: Anti-CD20 IFL, linker; Amino acid: GGGGSGGGGSGGGGS

[0130] Accession No. 7: Anti-CD20 IFL, immunoglobulin variable domain of rituximab heavy chain; cDNA:

Chem.

[0131] Accession No. 8: Anti-CD20 IFL, immunoglobulin variable domain of rituximab heavy chain; Amino acid:

Chem.

[0132] Accession No. 9: anti-CD20 IFL, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; cDNA:

Chemical Structure

[0133] Accession No. 10: anti-CD20 IFL, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; amino acids:

Chemical Structure

[0134] Accession No. 11: anti-CD19 IFL, CD8α signal peptide; cDNA: ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0135] Accession No. 12: anti-CD19 IFL, CD8α signal peptide; amino acids: MALPVTALLLPLALLLHAARP

[0136] Accession No. 13: anti-CD19 IFL, immunoglobulin variable domain of the light chain; cDNA:

Chemical Structure

[0137] Accession No. 14: anti-CD19 IFL, immunoglobulin variable domain of the light chain; amino acids: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0138] Accession No. 15: anti-CD19 IFL, linker; cDNA: GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT

[0139] Accession No. 16: anti-CD19 IFL, linker; amino acid: GGGGSGGGGSGGGGS

[0140] Accession No. 17: anti-CD19 IFL, immunoglobulin variable domain of heavy chain; cDNA:

Chemical formula

[0141] Accession No. 18: anti-CD19 IFL, immunoglobulin variable domain of heavy chain; amino acid:

Chemical formula

[0142] Accession No. 19: anti-CD19 IFL, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; cDNA:

Chemical formula

[0143] Accession No. 20: anti-CD19 IFL, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; amino acid:

Chemical formula

[0144] Accession No. 21: anti-CD20 IFL-P2A-CD16V-BB-ζ, rituximab signal peptide; cDNA: ATGGATTTCCAGGTCCAGATTATTTCCTTCCTGCTGATTAGTGCCAGT

[0145] Accession No. 22: Anti-CD20 IFL-P2A-CD16V-BB-ζ, rituximab signal peptide; Amino acids: MDFQVQIISFLLISAS

[0146] Accession No. 23: Anti-CD20 IFL-P2A-CD16V-BB-ζ, immunoglobulin variable domain of rituximab light chain; cDNA:

Chem.

[0147] Accession No. 24: Anti-CD20 IFL-P2A-CD16V-BB-ζ, immunoglobulin variable domain of rituximab light chain; Amino acids: VIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0148] Accession No. 25: Anti-CD20 IFL-P2A-CD16V-BB-ζ, linker; cDNA: GGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGCTCC

[0149] Accession No. 26: Anti-CD20 IFL-P2A-CD16V-BB-ζ, linker; Amino acids: GGGGSGGGGSGGGGS

[0150] Accession No. 27: Anti-CD20 IFL-P2A-CD16V-BB-ζ, immunoglobulin variable domain of rituximab heavy chain; cDNA:

Chem.

[0151] Accession No. 28: Anti-CD20 IFL-P2A-CD16V-BB-ζ, immunoglobulin variable domain of rituximab heavy chain; Amino acids:

Chem.

[0152] Accession No. 29: Anti-CD20 IFL-P2A-CD16V-BB-ζ, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; cDNA:

Chem.

[0153] Accession No. 30: Anti-CD20 IFL-P2A-CD16V-BB-ζ, hinge and constant heavy domains 2 and 3 of immunoglobulin G1; Amino acid:

Chem.

[0154] Accession No. 31: Anti-CD20 IFL-P2A-CD16V-BB-ζ, P2A; cDNA: GCCACAAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGA

[0155] Accession No. 32: Anti-CD20 IFL-P2A-CD16V-BB-ζ, P2A; Amino acid: ATNFSLLKQAGDVEENPG

[0156] Accession No. 33: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD8α signal peptide; cDNA: CCCGCCCTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCCCGCCCA

[0157] Accession No. 34: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD8α signal peptide; Amino acid: PALPVTALLLPLALLLHAARP

[0158] Accession No. 35: Anti-CD20 IFL-P2A-CD16V-BB-ζ, extracellular domain of FCGR3A; cDNA:

Chem.

[0159] SEQ ID NO: 36: Anti-CD20 IFL-P2A-CD16V-BB-ζ, FCGR3A extracellular domain; Amino acids:

Chem.

[0160] SEQ ID NO: 37: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD8α hinge and transmembrane; cDNA:

Chem.

[0161] SEQ ID NO: 38: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD8α hinge and transmembrane; Amino acids: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0162] SEQ ID NO: 39: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD137 cytoplasmic domain; cDNA:

Chem.

[0163] SEQ ID NO: 40: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD137 cytoplasmic domain; Amino acids: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0164] SEQ ID NO: 41: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD3ζ intracellular domain; cDNA:

Chem.

[0165] Accession number 42: Anti-CD20 IFL-P2A-CD16V-BB-ζ, CD3ζ cytoplasmic domain; Amino acids:

Chem.

[0166] Accession number 43: P2A cDNA: GCCACAAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGA

[0167] Accession number 44: P2A amino acids: ATNFSLLKQAGDVEENPG

[0168] Accession number 45: T2A cDNA: GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCT

[0169] Accession number: 46: T2A amino acids: GSGEGRGSLLTCGDVEENPGP

[0170] Accession number 47: E2A cDNA: GGAAGCGGACAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAACCCTGGACCT

[0171] Accession number 48: E2A amino acids: GSGQCTNYALLKLAGDVESNPGP

[0172] Accession number 49: F2A cDNA: GGAAGCGGAGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCT

[0173] Accession number: 50: F2A amino acids: GSGVKQTLNFDLLKLAGDVESNPGP

[0174] SEQ ID NO: 51: Linker Nucleotides of Figure 8C: AGCTGCTGCTAAGGCACTGGAAGCAGAAGCCGCGGCTAAGGAGGCGGCTGCAAAAGAAGCTGCAGCCAAGGAAGCAGCCGCGAAGGCA

[0175] SEQ ID NO: 52: Linker Amino Acids of Figure 8C: AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA

[0176] SEQ ID NO: 53: IL-15 Nucleotides of Figure 8C:

Chem.

[0177] SEQ ID NO: 54: IL-15 Amino Acids of Figure 8C: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0178] Incorporation by Reference; Equivalents The teachings of all patents, published applications, and references cited herein are hereby incorporated by reference in their entirety.

[0179] Although the exemplary embodiments have been shown and described in detail, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. Examples of the present invention include the following. Item 1 An immune cell expressing a peptide, wherein the peptide a) a single-chain variable fragment (scFv) domain; b) a crystallizable fragment (Fc) domain; and c) a hinge domain connecting the scFv domain and the Fc domain An immune cell comprising. Item 2 The scFv domain is an immunoglobulin variable light (V L ) domain, an immunoglobulin variable heavy (V H ) domain, and a linker domain connecting the V L and V H domains, the immune cell according to item 1. Item 3 The linker domain is (G 4 S) x where x is an integer from 1 to 100, the immune cell according to item 2. Item 4 The linker domain is (G 4 S) 3 The immune cell according to item 3. Item 5 The scFv domain binds to CD19, the immune cell according to any one of items 1 to 4. Item 6 The scFv domain binds to CD20, the immune cell according to any one of items 1 to 4. Item 7 The scFv domain binds to CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80, or CD86, the immune cell according to any one of items 1 to 4. Item 8 The Fc domain is an immunoglobulin constant heavy 2 (C H 2) domain and an immunoglobulin constant heavy 3 (C H 3) domain, the immune cell according to any one of items 1 to 4. Item 9 The Fc domain is a human IgG1 Fc domain, the immune cell according to any one of items 1 to 4. Item 10 The peptide further comprises a signal peptide at the N-terminus relative to the scFv domain, the immune cell according to any one of items 1 to 4. Item 11 The peptide further comprises a self-cleaving peptide that connects the Fc domain to a chimeric receptor, the chimeric receptor comprising a receptor domain, a hinge and transmembrane domain, a co-stimulatory signaling domain, and a cytoplasmic signaling domain, the immune cell according to any one of items 1 to 4. Item 12 The self-cleaving peptide is a 2A peptide, the immune cell according to item 11. Item 13 The immune cell according to item 11, wherein the receptor domain is CD16. Item 14 The immune cell according to item 11, wherein the hinge and transmembrane domains are the CD8α hinge and transmembrane domains. Item 15 The immune cell according to item 11, wherein the co-stimulatory domain is the 4-1BB co-stimulatory domain. Item 16 The immune cell according to item 11, wherein the cytoplasmic signaling domain is CD3ζ cytoplasmic signaling. Item 17 The immune cell according to item 11, wherein the chimeric receptor is CD16V-4-1BB-CD3ζ. Item 18 The scFv domain binds to CD19 or CD20, the Fc domain is a human IgG1 Fc domain, and the hinge domain is an IgG1 hinge domain; the peptide further comprises a CD8α signal peptide at the N-terminus relative to the scFv domain; the peptide further comprises a chimeric receptor that is CD16V-4-1BB-CD3ζ, the immune cell according to any one of items 1 to 4. Item 19 The immune cell according to any one of items 1 to 4, wherein the peptide further comprises one or more of the following mutations: S239D; S267E; H268F; or I332E. Item 20 The immune cell according to any one of items 1 to 4, wherein the peptide further comprises one or more of the following mutations: E345K; E430G; or S440Y. Item 21 The immune cell according to any one of items 1 to 4, wherein the peptide further comprises IL-15 linked to the Fc domain by a linker. Item 22 The linker that links IL-15 to the Fc domain is SEQ ID NO: 51; A(EAAK) 4 ALEA(EAAAK) 4 A;(EAAAK) z ;A(EAAAK) z A; and (XP) w selected from the group consisting of, z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100, the immune cell according to item 21. Item 23 The immune cell according to any one of items 1 to 4, wherein the peptide further comprises a ligand that binds to 4-1BB (CD37), CD28, or OX40 (CD134) linked to the Fc domain by a linker. Item 24 The linker that links IL-15 to the Fc domain is SEQ ID NO: 51; A(EAAK) 4 ALEA(EAAAK) 4 A;(EAAAK) z ;A(EAAAK) z A; and (XP) w The immune cell according to item 23, wherein z is an integer from 1 to 100 selected from the group consisting of; X is any amino acid, and w is an integer from 1 to 100. Item 25 The immune cell according to any one of items 1 to 4, wherein the immune cell is a natural killer cell. Item 26 The immune cell according to any one of items 1 to 4, wherein the immune cell is a T lymphocyte cell.

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Claims

1. A T lymphocyte cell or natural killer cell that expresses a peptide, wherein the peptide comprises: a) a single-chain variable fragment (scFv) domain, wherein the scFv domain comprises: i) Immunoglobulin variable light (V Ｌ ) domain, ii) Immunoglobulin variable heavy (V Ｈ ) domain, and iii) said V Ｌ and V Ｈ a linker domain connecting the domains, wherein the linker domain connecting the VL and VH domains is (G4S)3 ; b) comprising SEQ ID NO: 10 and having, C-terminal to said scFv domain, the hinge of IgG1 and constant heavy 2 (C Ｈ 2) domain and constant heavy 3 (C Ｈ 3) domain c) a CD8α signal peptide N-terminal to the scFv domain; and d) The constant heavy 3 (C Ｈ 3) domain is at the C-terminus with respect to the IgG1 and is a linker domain connecting the IL-15 domain A T lymphocyte cell or natural killer cell comprising.

2. The T lymphocyte cell or natural killer cell according to claim 1, wherein the scFv domain binds to CD19.

3. The T lymphocyte cell or natural killer cell according to claim 1, wherein the scFv domain binds to CD20.

4. The T lymphocyte cell or natural killer cell according to claim 1, wherein the scFv domain binds to CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80, or CD86.

5. The T lymphocyte cell or natural killer cell according to claim 1, wherein the peptide further comprises a self-cleaving peptide that links to a chimeric receptor, and the chimeric receptor comprises a receptor domain, a hinge and transmembrane domain, a co-stimulatory signaling domain, and a cytoplasmic signaling domain.

6. The T lymphocyte cell or natural killer cell according to claim 5, wherein the self-cleaving peptide is a 2A peptide.

7. The T lymphocyte cell or natural killer cell according to claim 5, wherein the receptor domain is a CD16 receptor domain.

8. The T lymphocyte cell or natural killer cell according to claim 5, wherein the hinge and transmembrane domain is a CD8α hinge and transmembrane domain.

9. The T lymphocyte cell or natural killer cell according to claim 5, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

10. The T lymphocyte cell or natural killer cell according to claim 5, wherein the cytoplasmic signaling domain is a CD3ζ cytoplasmic signaling domain.

11. The T lymphocyte cell or natural killer cell according to claim 5, wherein the chimeric receptor is CD16V-4-1BB-CD3ζ.

12. The linker domain at the C-terminus with respect to the constant heavy 3 (C Ｈ 3) domain of the IgG1 is selected from the group consisting of SEQ ID NO: 52; and A(EAAA ｚ A, where z is an integer from 2 to 5. The T lymphocyte cell or natural killer cell according to claim 1.

13. The T lymphocyte cell or natural killer cell according to claim 1, wherein the peptide further comprises a ligand that binds to 4-1BB (CD37), CD28, or OX40 (CD134), which are linked by a linker.

14. The linker is selected from the group consisting of SEQ ID NO: 52; and A (EAAAK) ｚ The T lymphocyte cell or natural killer cell according to claim 13, wherein A is selected from the group consisting of A, and z is an integer of 2 to 5.

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