Neutralization of Human Cytokines with Membrane-Bound Anti-Cytokine Non-Signaling Binders Expressed in Immune Cells

Abstract

Transgenic T cells and vectors for making transgenic T cells are described. The vectors can include a nucleic acid encoding a membrane-bound anti-IL6 (mb-alL6) single chain variable fragment (scFv), and the transgenic T cells can express mb-alL6. The transgenic T cells are useful for suppressing proliferation of IL-6-dependent cells, reducing IL-6 concentration, or both. In one embodiment, the vector is a bicistronic construct encoding the mb-alL6 and an anti-CD19-41 BB-003 chimeric antigen receptor (CAR). In another embodiment, an anti-IL-6 scFv can be linked to a 41 BB and 003 domains to form an anti-IL-6 CAR. The transgenic T cells expressing said constructs can reduce the linker risk of cytokine release syndrome (CRS) in cancer patients being treated with CAR T cell or for the treatment of autoimmune diseases and inflammatory diseases in which cytokines are involved in pathogenesis.

Claims

1. A vector comprising a nucleic acid encoding a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv), the mb-aIL6 scFv comprising: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

2. The vector of claim 1, wherein one or more of the anti-IL-6 variable light chain domain and the anti-IL-6 variable heavy chain domain are human anti-IL6 variable light and variable heavy chains domains.

3. The vector of claim 2, wherein the variable light chain domain has at least 90% sequence identity to SEQ ID NO: 4.

4. The vector of claim 2, wherein the variable heavy chain domain has at least 90% sequence identity to SEQ ID NO: 8.

5. The vector of claim 1, wherein the linker domain is (G4S)x, wherein x is an integer from 1 to 100.

6. The vector of claim 1, wherein the linker domain is (G4S)3.

7. The vector of claim 1, wherein the hinge and transmembrane domain are CD8 hinge and transmembrane domain.

8. The vector of claim 1, wherein the nucleic acid further encodes a chimeric antigen receptor (CAR).

9. The vector of claim 8, wherein the chimeric antigen receptor is an anti-CD19-41BB-CD3 chimeric antigen receptor (CAR).

10. The vector of claim 9, wherein the mb-aIL6 is coupled to the anti-CD19-41BB-CD3 by a P2A sequence.

11. A vector comprising a nucleic acid encoding an anti-IL6 chimeric antigen receptor (CAR), the anti-IL6 CAR comprising: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a first linker domain joining the variable light chain domain and the variable heavy chain domain; b) a second linker domain N-terminal to the anti-IL6 scFv; c) a hinge and transmembrane domain N-terminal to the second linker domain; and d) an intracellular signaling domain N-terminal to the hinge and transmembrane domain.

12. The vector of claim 11, wherein the intracellular signaling domain is a 41BB domain.

13. The vector of claim 11, further comprising a co-stimulatory domain N-terminal to the intracellular signaling domain.

14. The vector of claim 13, wherein the co-stimulatory domain is a CD3 domain.

15. A method of reducing IL-6 concentration in a mammal, the method comprising: expressing in a T cell a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv) and contacting the T cell with a fluid containing IL-6, wherein the mb-aIL6 comprises: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

16. The method of claim 15, wherein the mammal is a human.

17. The method of claim 15, further comprising culturing the T cell to generate new T cells that express the mb-aIL6.

18. The method of claim 15, further comprising reducing risk of cytokine release syndrome (CRS), and wherein the T cells of the mammal express a chimeric antigen receptor.

19. The method of claim 15, wherein the mammal is suffering from an autoimmune disease, an inflammatory disease, or a lymphoproliferative disorder, and wherein reducing IL-6 concentration in the mammal treats the autoimmune disease, the inflammatory disease, or the lymphoproliferative disorder, respectively.

20. The method of claim 15, wherein the mammal is suffering from rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, or Castleman disease, and wherein reducing IL-6 concentration treats rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, or Castleman disease, respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0013] FIGS. 1A-D pertain to design and expression of mb-aIL6. FIG. 1A is a schema of the mb-aIL6 construct. FIG. 1B is a schema of the MSCV-mb-aIL6-IRES-GFP plasmid. FIG. 1C is flow cytometric analysis of Jurkat cells transduced with either GFP alone (Mock) or GFP plus mb-aIL6. Dot plots illustrate GFP fluorescence, and mb-aIL6 expression after staining with biotin-conjugated goat anti-human F(ab)2 antibody and streptavidin-APC (Jackson ImmunoResearch Laboratories). FIG. 1D is flow cytometric analysis of IL-6 binding to mock- or mb-aIL6-transduced Jurkat cells. Dot plots illustrate GFP fluorescence, and IL-6 binding after staining with IL-6 biotin (Abcam) and streptavidin-APC. Soybean-trypsin inhibitor (STI)-biotin was used as a control.

[0014] FIGS. 2A-E pertain to functionality of the mb-aIL6 construct. FIG. 2A is a chart showing levels of IL-6. Jurkat cells (210.sup.6/mL) transduced with either GFP alone (Mock) or GFP plus mb-aIL6 were cultured for 2 hours in tissue culture medium containing 1 ng/mL human IL-6. At the end of the cultures, levels of IL-6 were measured by ELISA. FIG. 2B is a graph showing that IL-6 neutralization is cell-dose dependent. Different cell concentrations were tested (0.25-210.sup.6/mL) and levels of IL-6 in the supernatant after 2 hours of culture were measured by ELISA. FIG. 2C is a graph showing that IL-6 neutralization is time-dependent. Cultures were set up as described with respect to FIG. 2A and levels of IL-6 in the supernatant were measured after the indicated culture times. Cells were incubated for 10, 30, 60, and 120 minutes. Dashed curve represents fitted exponential decay curve. FIG. 2D is a chart showing that mb-aIL6 Jurkat cells remained effective at low IL-6 concentrations. Cultures were set up as described with respect to FIG. 2A but with initial concentrations of IL-6 in the tissue culture medium ranging from 0.025 to 0.2 ng/mL and Jurkat cells at 0.510.sup.6/mL. FIG. 2E is a graph showing that IL-6 neutralization is augmented by allowing proliferation of Jurkat-mb-aIL6 cells. Cultures were set up as described with respect to FIG. 2A but with an initial cell concentration of 0.210.sup.6 cells/mL; IL-6 in the supernatant was measured by ELISA after 2-72 hours.

[0015] FIGS. 3A-B show that cells expressing mb-aIL6 abrogate IL-6-dependent signal transduction and cell proliferation. FIG. 3A is a chart showing Stat3 phosphorylation in U937 cells triggered by IL-6 is prevented by exposure to mb-aIL6 Jurkat cells. Jurkat cells (210.sup.6/mL) untransduced (WT) or transduced with GFP plus mb-aIL6 were cultured for 2 hours in tissue culture medium containing 1 ng/mL human IL-6. The supernatant was then added to U937 cells for 15 minutes at 37 C. Tissue culture medium (no T cells) with or without IL-6 was used as a control. Bars show the mean (SD) of Stat3 phosphorylation as measured by flow cytometry after staining with an anti-PhosphoStat3 antibody (BD Biosciences anti-Stat3 pY705). ***P<0.001. FIG. 3B is a graph showing the IL-6-dependent proliferation of DS-1 cells is suppressed by exposure to mb-aIL6 Jurkat cells. DS-1 transduced with mCherry and either mock-transduced or mb-IL6 transduced Jurkat cells were co-cultured at a 1:1 ratio, with IL-6 (0.5 ng/mL). DS-1 proliferation was quantitated using the IncuCyte live Imaging System (Essen); results are expressed as mean (SD) of red calibrated unit (RCU)m.sup.2/well in triplicate measurements. ** P<0.01 for the measurements at 120 hours.

[0016] FIGS. 4A-D show expression and functionality of the mb-aIL6 construct on peripheral blood T cells. FIG. 4A is a flow cytometric analysis of peripheral blood T cells transduced with either GFP alone (Mock) or GFP plus mb-aIL6. Dot plots illustrate GFP fluorescence, and mb-aIL6 expression after staining with biotin-conjugated goat anti-human F(ab)2 antibody and streptavidin-APC. FIG. 4B is a flow cytometric analysis of IL-6 binding to mock- or mb-aIL6-transduced peripheral blood T cells. Dot plots illustrate GFP fluorescence, and IL-6 binding after staining with IL-6 biotin and streptavidin-APC. FIG. 4C is a cell marker profile of mock- or mb-aIL6-transduced peripheral blood T cells, labelled with anti-CD3 APC, anti-CD56 PE, anti-CD4 V450, and anti-CD8 PerCP (BD Biosciences). FIG. 4D is a graph showing the IL-6-dependent proliferation of DS-1 cells is suppressed by exposure to mb-aIL6 T lymphocytes. DS-1 transduced with mCherry and either mock-transduced or mb-aIL6 transduced T cells were co-cultured at a 1:1 ratio, with IL-6 (0.5 ng/mL). DS-1 proliferation was quantitated using the IncuCyte live Imaging System (Essen); results are expressed as mean (SD) of red calibrated unit (RCU)m.sup.2/well in triplicate measurements. ** P<0.01 for the measurements at 120 hours.

[0017] FIGS. 5A-C show the design, expression, and IL-6 neutralizing capacity of a bicistronic construct encoding mb-aIL6 and anti-CD19 CAR. FIG. 5A is a schema of the MSCV plasmid containing both receptors (DUAL). FIG. 5B is a flow cytometric analysis of peripheral blood T-cells transduced with either GFP alone (Mock), GFP plus anti-CD19-41BB-CD3 CAR, mb-aIL6, or both. Dot plots illustrate mb-aIL6 expression after staining with biotin-conjugated goat anti-human F(ab)2 antibody and streptavidin-APC, and anti-CD19 CAR expression after staining with CD19-myc, followed by R-phycoerythrin (PE)-conjugated anti-myc (Cell Signaling Technology). FIG. 5C is a graph showing neutralization of IL-6 by mb-aIL6 T lymphocytes is not affected by CAR co-expression. T lymphocytes transduced as described with respect to FIG. 5B were cultured in tissue culture medium containing 1 ng/mL IL-6. After 2 hours, IL-6 was measured in the supernatant by ELISA.

[0018] FIGS. 6A-D show expression of mb-aIL6 does not affect anti-CD19 CAR function. FIG. 6A is a plot showing IFN-7 production in T-cells co-cultured with CD19+ ALL cell line OP-1 for 6 hours at an E:T ratio of 1:1. Expression of IFN was measured by flow cytometry after staining with PE-conjugated anti-human IFN antibody (BD Biosciences). Symbols represent results of triplicate experiments obtained with T cells from 3 donors. FIG. 6B is a plot showing CD107a expression in T-cells co-cultured with OP-1 for 4 hours at an E:T ratio of 1:1. CD107a expression was measured by flow cytometry after staining with PE-conjugated anti-human CD107a antibody (BD Biosciences). FIG. 6C is a plot showing cytotoxicity of T-cells against OP-1 after 4 hours at an E:T ratio of 1:1. FIG. 6D is two charts showing proliferation of T cells co-cultured with or without irradiated OP-1 for 21 days at an E:T ratio of 1:1 with 120 IU/mL IL-2. Irradiated OP-1 cells were added on day 0, day 7 and day 14. Symbols represent the mean (SD) of triplicate measurements.

[0019] FIGS. 7A-B show the function of T cell expressing mb-aIL6 and anti-CD19 CAR in an in vitro model of CRS. FIG. 7A is two graphs showing cytotoxicity of T-cells co-cultured with mCherry-transduced OP-1 cells with or without THP-1 cells (1:5:1 T-cell:OP-1:THP-1 ratio). OP-1 cell numbers were quantitated using the IncuCyte Live Imaging System (Essen); results are expressed as mean (SD) of red calibrated unit (RCU)m.sup.2/well in triplicate measurements. FIG. 7B is a chart showing levels of IL-6 in the supernatant of the cultures shown in FIG. 7A after 40 hours of culture, measured by ELISA.

[0020] FIG. 8A-C are schematics of other IL-6 neutralizing receptors. FIG. 8A is a schematic of a nucleic acid construct for secreted aIL6 and a cell expressing secreted aIL6. FIG. 8B is a schematic of a nucleic acid construct for an anti-IL-6 scFv linked to a 41BB domain and a CD3 domain, forming an anti-IL6 CAR. FIG. 8C is a schematic of a nucleic acid construct where the anti-IL6 scFv is replaced by an IL-6 receptor deprived of signaling capacity.

[0021] FIGS. 9A-B show expression of mb-aIL6 in T cells neutralizes IL-6 in vivo. NOD.Cg-Prkdc.sup.scid IL2rg.sup.tm1Wj1/SzJ (NOD/scid IL2RGnull) mice (Jackson Laboratory, Bar Harbor, Me.) were injected intraperitoneally with 110.sup.6 DS-1 cells on day 0, and 110.sup.7 peripheral blood T-cells transduced with either GFP alone (Mock) or GFP plus mb-aIL6 on day 2. Tumor engraftment and growth was measured using the Xenogen IVIS-200 system (Caliper Life Sciences). All mice received 1000 IU human IL-6 and 20000 IU human IL-2 every 2 days intraperitoneally starting from day 0. FIG. 9A is ventral and dorsal images of mice. FIG. 9B is a chart showing change in luminescence in mice engrafted with DS-1 expressing luciferase. Each symbol corresponds to one bioluminescence measurement; lines connect all measurements in one mouse.

[0022] FIGS. 10A-F show that expression of mb-aIL6 does not affect anti-CD19 CAR function in vivo. NOD.Cg-Prkdc.sup.scid IL2rg.sup.tm1Wj1/SzJ (NOD/scid IL2RGnull) mice (Jackson Laboratory, Bar Harbor, Me.) were injected intravenously with 0.510.sup.6 Nalm-6 cells on day 0, and 210.sup.7 peripheral blood T-cells transduced with either anti-CD19-41BB-CD3 (CAR) or the CAR plus mb-aL6 (CAR+mb-aIL6) on day 3. Tumor engraftment and growth was measured using the Xenogen IVIS-200 system (Caliper Life Sciences). All mice received 20000 IU human IL-2 every 2 days intraperitoneally starting from day 0. Mice were euthanized when the signal threshold reached 10.sup.10 photons/sec. FIG. 10A is ventral and dorsal images of mice. Images on day 3 were processed with enhanced sensitivity to show the presence of tumors before injection of engineered T-cells. FIG. 10B is a chart showing change in luminescence in mice engrafted with Nalm-6 expressing luciferase. FIG. 10C is ventral and dorsal images of mice. Images on day 3 were processed with enhanced sensitivity to show the presence of tumors before injection of engineered T-cells. FIG. 10D is a chart showing CAR T-cell counts on day 53. Blood from mice were obtained via cheek prick and CAR T-cells were quantified using flow cytometry. FIG. 10E is a chart showing the survival curve of the mice. Curves for mice injected with no T cells versus those injected with CAR-T cells or CAR-T+mb-aIL6 was calculated by log rank test (P<0.01 for either comparison). FIG. 10F is a chart showing change in luminescence in mice engrafted with Nalm-6 expressing luciferase. Each symbol corresponds to one bioluminescence measurement; lines connect all measurements in one mouse.

[0023] FIGS. 11A-B pertain to design of mb-aTNF. FIG. 11A is a schema of the mb-aTNF construct. FIG. 11B is a schema of the MSCV-mb-aTNF-IRES-GFP plasmid.

DETAILED DESCRIPTION

[0024] A description of example embodiments follows.

Interleukin-6 and Cytokine Release Syndrome

[0025] Interleukin-6 (IL-6) is a pro-inflammatory cytokine that is involved in the pathogenesis of multiple autoimmune diseases, inflammatory diseases, and lymphoproliferative disorders, including graft-versus-host disease (GvHD), rheumatoid arthritis, and systemic lupus erythematosus. IL-6 is also involved in the development of cytokine release syndrome (CRS), one of the most common side effects of infusion of T cells redirected with chimeric antigen receptors (CARs). CRS can be severe and, in some cases, fatal..sup.7-10 IL-6 is central to the development of CRS, and an anti-IL-6 receptor antibody (tocilizumab) is currently used to curb its effects..sup.7-10

[0026] The vectors described herein can be used to generate modified T cells, which, in turn, can be used for treatment of auto-immune diseases and CRS. The processes described herein can be used to create transgenic T cells that can neutralize IL-6, thereby decreasing the risk and/or severity of CRS. While the particular examples described herein use IL-6 as a paradigm, the approach is applicable to the neutralization of other cytokines involved in the pathogenesis of autoimmune diseases and CRS.

Nucleic Acids

[0027] As used herein, the term nucleic acid refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). Nucleic acid includes, 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 either or both strands of the molecule.

[0028] The terms nucleotide and nucleotide monomer refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, 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.

[0029] As used herein, the term sequence identity, refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which a test sequences are compared. The sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence.

[0030] Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, the alignment can include introduced gaps to provide for the maximal 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 search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized 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).

[0031] When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through 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)).

[0032] In various embodiments, two nucleotide sequences, or two amino acid sequences, can have at least, e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity. When ascertaining percent sequence identity to one or more sequences described herein, the sequences described herein are the reference sequences.

[0033] In some embodiments, the variable light chain domain has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to SEQ ID NO: 4. In some embodiments, the variable heavy chain domain has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to SEQ ID NO: 8.

Vectors

[0034] The terms vector, vector construct and expression vector mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a plasmid, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid 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 allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an expression product such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be expressed by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when 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.

[0036] Gene delivery vectors generally include a transgene (e.g., nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in the host cells into which the vector is introduced. Suitable promoters for gene expression and delivery constructs are known in the art. Recombinant plasmids can also comprise inducible, or regulatable, promoters for expression of an enzyme in cells.

[0037] Various gene delivery vehicles are known in the art and include both 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, e.g., vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector (AAV), and murine stem cell virus (MSCV). The viral vector can be replicating or non-replicating. Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.

[0038] Non-viral vectors for gene delivery include naked DNA, plasmids, transposons, and mRNA, among others. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), pMAL plasmids (New England Biolabs, Beverly, Mass.). Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.

[0039] In certain embodiments, the vector comprises an internal ribosome entry site (IRES). In some embodiments, the vector includes a selection marker, such as an ampicillin resistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein, such as green fluorescent protein (GFP). 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 the insertion of the desired gene.

[0040] Although the genetic code is degenerate in that most amino acids are represented by multiple codons (called synonyms or synonymous codons), it is understood in the art that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. Accordingly, in some embodiments, the vector includes a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g., through codon optimization). Codon optimization refers to a process in which a polynucleotide encoding a protein of interest is modified to replace particular codons in that polynucleotide with codons that encode the same amino acid(s), but are more 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.

Membrane-Bound Anti-Cytokine Constructs

[0041] Membrane-bound anti-cytokine constructs, an example of which is the mb-aIL6 construct of FIG. 1A, can be created as described herein. The anti-cytokine construct can be specific to a wide variety of cytokines, such as IL-6, (TNF)-, IL-1, IL-12, IL-17, IL-18, IFN, and others.

[0042] FIG. 1A illustrates a particular construct that is an anti-IL6 single-chain variable fragment (anti-IL6 scFv) coupled to a hinge and transmembrane domain. The anti-IL6 scFv includes an anti IL-6 variable light chain domain, an anti IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain. The relative positions of the variable light and variable heavy chain domain can be reversed, but they are both N terminal to a transmembrane domain, illustrated in FIG. 1A as a CD8 hinge and transmembrane domain. The construct can also include an N-terminal signal peptide (not shown in FIG. 1A), such as a CD8 signal peptide. FIG. 1B is a schema for a MSCV mb-aIL6-IRES-GFP plasmid.

[0043] A variety of linker domains are suitable. In some embodiments, the linker domain can be (G4S).sub.x, wherein x is an integer from 1 to 100. In some embodiments, the linker domain can be (G4S).sub.3. In other embodiments, the linker domain can be one or more glycine residues. In other embodiments, the linker domain can be (EAAAK).sub.3.

[0044] A variety of hinge and transmembrane domains are suitable. In some embodiments, the hinge domain can be a CD8 hinge domain. In some embodiments, the transmembrane domain can be a CD8 transmembrane domain. In some embodiments, the hinge and transmembrane domain can be a CD8 hinge and transmembrane domain. In some embodiments, the hinge can be a plurality of glycine and serine residues. In some embodiments, the transmembrane domain can be a transmembrane domain from CD4, CD8, CD16, CD28, CD32, CD34, CD64, CD137, FcRI, OX40, CD3, CD3, CD3, CD3, TCR, VEGFR2, FAS, or FGFR2B.

[0045] While the embodiment of FIG. 1A is an anti-IL6 construct, a similar approach can be applied to generate constructs for other cytokines, such as tumor necrosis factor (TNF)-, IL-1, IL-12, IL-17, IL-18, IFN, etc., and/or block their receptors. For example, based on the schema in FIG. 1A, the anti-IL6 scFv portion can be replaced with a different scFv that specifically binds to a different cytokine, such as (TNF)- (FIG. 11), IL-1, IL-12, IL-17, IL-18, or IFN. All of the teachings herein are equally applicable to constructs for expression of a membrane-bound protein that neutralizes cytokines.

[0046] Multiple neutralizing receptors can be expressed on the same cell or in different cell subsets to exert a comprehensive and long-lasting anti-inflammatory effect.

Methods of Making Transgenic Host Cells

[0047] Described herein are methods of making a transgenic host cell, such as transgenic T cells. The transgenic host cells can be made, for example, by introducing one or more of the vector embodiments described herein into the host cell.

[0048] In one embodiment, the method comprises introducing into a host cell a vector that includes a nucleic acid that encodes a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv). In some embodiments, the nucleic acid of the vector can further encode a chimeric antigen receptor (CAR), such as an anti-CD19-41BB-CD3. In some embodiments, a nucleic acid, such as a bicistronic vector, expresses the mb-aIL6 and the CAR. In some embodiments, two separate vectors can be used to create a transgenic cell, such as a transgenic T cell, that expresses mb-aIL6 and the CAR.

[0049] In some embodiments, one or more of the nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acids to be integrated into a host genome can be introduced into the host cell using any of a variety of suitable methodologies known in the art, including, for example, homologous recombination, CRISPR-based systems (e.g., CRISPR/Cas9; CRISPR/Cpf1) and TALEN systems.

Values and Ranges

[0050] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in various embodiments, unless the context clearly dictates otherwise. About in reference to a numerical value generally refers to a range of values that fall within 8%, in some embodiments 6%, in some embodiments 4%, in some embodiments 2%, in some embodiments 1%, in some embodiments 0.5% of the value unless otherwise stated or otherwise evident from the context.

Example Embodiments: Vector

[0051] An embodiment is a vector that includes a nucleic acid encoding a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv). The mb-aIL6 scFv includes a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

[0052] In some embodiments, one or more of the anti-IL-6 variable light chain domain and the anti-IL-6 variable heavy chain domain are human anti-IL6 variable light and variable heavy chains domains. In some embodiments, the variable light chain domain has at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the variable heavy chain domain has at least 90% sequence identity to SEQ ID NO: 8.

[0053] In some embodiments, the linker domain is (G4S).sub.x, wherein x is an integer from 1 to 100. In some embodiments, the linker domain is (G4S).sub.3. In some embodiments, the linker domain is one or more glycine residues. In some embodiments, the linker domain is (EAAAK).sub.3.

[0054] In some embodiments, the hinge and transmembrane domain are CD8 hinge and transmembrane domain. In some embodiments, the hinge comprises a plurality of glycine and serine residues. In some embodiments, the transmembrane domain is a transmembrane domain from CD4, CD8, CD16, CD28, CD32, CD34, CD64, CD137, FcRI, OX40, CD3, CD3, CD3, CD3, TCR, VEGFR2, FAS, or FGFR2B.

[0055] In some embodiments, the nucleic acid further encodes a chimeric antigen receptor (CAR), such as an anti-CD19-41BB-CD3 chimeric antigen receptor (CAR). In some embodiments, the mb-aIL6 is coupled to the anti-CD19-41BB-CD3 by a P2A sequence.

Example Embodiments: Vector

[0056] Another embodiment is a vector that includes a nucleic acid encoding an anti-IL6 (aIL6) single-chain variable fragment (scFv). The aIL6 scFv includes: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain.

Example Embodiments: Vector

[0057] Another embodiment is a vector that includes a nucleic acid encoding an anti-IL6 chimeric antigen receptor (CAR). The anti-IL6 CAR includes: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a first linker domain joining the variable light chain domain and the variable heavy chain domain; b) a second linker domain N-terminal to the anti-IL6 scFv; c) a hinge and transmembrane domain N-terminal to the second linker domain; and d) an intracellular signaling domain N-terminal to the hinge and transmembrane domain.

[0058] In some embodiments, the intracellular signaling domain is a 41BB domain.

[0059] In some embodiments, the vector further includes a co-stimulatory domain N-terminal to the intracellular signaling domain. In some embodiments, the co-stimulatory domain is a CD3 domain.

Example Embodiments: Vector

[0060] Another embodiment is a vector that includes a nucleic acid encoding a membrane-bound IL-6 receptor. The membrane-bound IL-6 receptor includes: a) an extracellular domain; b) a linker domain N-terminal to the extracellular domain; c) an IL-6 receptor domain; and d) a transmembrane domain.

[0061] In some embodiments, the extracellular domain is a gp130 extracellular domain.

[0062] In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

Example Embodiments: Mammalian T Cell

[0063] Another embodiment is a mammalian T cell that includes a transgene encoding a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv), in accordance with any embodiment described herein.

[0064] In some embodiments, the mb-aIL6 scFv can include: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

[0065] In some embodiments, the mammalian T cell is a human T cell. In some embodiments, the mammalian T cell is a human peripheral blood T lymphocyte.

Example Embodiments: Method of Suppressing Proliferation of IL-6-Dependent Cells in a Mammal

[0066] Another embodiment is a method of suppressing proliferation of IL-6-dependent cells in a mammal. The method includes: expressing in a T cell a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv). The mb-aIL6 scFv can be in accordance with any embodiment described herein.

[0067] In some embodiments, the mb-aIL6 scFv includes: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

[0068] In some embodiments, the mammal is a human.

Example Embodiments: Method of Reducing IL-6 Concentration in a Mammal

[0069] Another embodiment is a method of reducing IL-6 concentration in a mammal. The method includes: expressing in a T cell a membrane-bound anti-IL6 (mb-aIL6) single-chain variable fragment (scFv) and contacting the T cell with a fluid containing IL-6. The mb-aIL6 scFv can be in accordance with any embodiment described herein.

[0070] In some embodiments, the mb-aIL6 includes: a) an anti-IL6 single-chain variable fragment (anti-IL6 scFv) comprising an anti-IL-6 variable light chain domain, an anti-IL-6 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-IL6 scFv.

[0071] In some embodiments, the mammal is a human.

[0072] In some embodiments, the method further includes culturing the T cell to generate new T cells that express the mb-aIL6.

[0073] In some embodiments, the method further includes reducing risk of cytokine release syndrome (CRS), and wherein the T cells of the mammal express a chimeric antigen receptor.

[0074] In some embodiments, the chimeric antigen receptor is an anti-CD19-41BB-CD3 CAR.

[0075] In some embodiments, the chimeric antigen receptor includes an anti-CD22 domain, an anti-CD20 domain, an anti-CD123 domain, a B-cell maturation antigen (BCMA) domain, an anti-mesothelin domain, an anti-CD7 domain, an anti-CD2 domain, an anti-CD5 domain, an anti-CD3 domain, an anti-Lewis Y domain, an anti-EpCam domain, an anti-Her2 domain, an or anti-prostate-specific membrane antigen (PSMA).

[0076] In some embodiments, the CAR further includes a 4-1BB domain, a CD3 domain, a CD28 domain, an inducible T cell co-stimulator (ICOS) domain, a DNAX-activating protein 10 (DAP10) domain, or a DNAX-activating protein 12 (DAP12) domain.

[0077] In some embodiments, the mammal is suffering from an autoimmune disease, and reducing IL-6 concentration in the mammal treats the autoimmune disease.

[0078] In some embodiments, the mammal is suffering from rheumatoid arthritis, and reducing IL-6 concentration treats rheumatoid arthritis.

[0079] In some embodiments, the mammal is suffering from systemic lupus erythematosus, and reducing IL-6 concentration treats systemic lupus.

[0080] In some embodiments, the mammal is suffering from an inflammatory disease, and reducing IL-6 concentration in the mammal treats the inflammatory disease.

[0081] In some embodiments, the mammal is suffering from graft-versus-host disease, and reducing IL-6 concentration treats graft-versus-host disease.

[0082] In some embodiments, the mammal is suffering from a lymphoproliferative disorder, and reducing IL-6 concentration treats the lymphoproliferative disorder.

[0083] In some embodiments, the mammal is suffering from Castleman disease, and reducing IL-6 concentration treats Castleman disease.

Example Embodiments: Vector

[0084] In another embodiment, a vector includes a nucleic acid encoding a membrane-bound anti-cytokine single-chain variable fragment (scFv). The membrane-bound anti-cytokine scFv can be in accordance with any embodiment described herein.

[0085] In some embodiments, the membrane-bound anti-cytokine includes: a) an anti-cytokine single-chain variable fragment (anti-cytokine scFv) comprising an anti-cytokine variable light chain domain, an anti-cytokine variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-cytokine scFv.

[0086] In some embodiments, the anti-cytokine is anti-(TNF)-, anti-IL-1, anti-IL-12, anti-IL-17, anti-IL-18, or anti-IFN.

EXEMPLIFICATION

Materials and Methods

Cells

[0087] Human cell lines Nalm-6 (B-cell acute lymphoblastic leukemia, ALL), Jurkat (T-cell ALL), THP-1 and U937 (acute monocytic leukemia), DS-1 (B-cell lymphoma) and HEK293T (embryonic kidney fibroblasts) were purchased from the American Type Culture Collection (Manassas, Va.). The B-ALL cell line OP-1 was developed in our laboratory..sup.11 Nalm-6, OP-1, THP-1, U937 and Jurkat cells were maintained in RPMI-1640 (ThermoFisher Scientific, Waltham, Mass.), supplemented with 10% fetal bovine serum (FBS) (HyClone GE Healthcare, Logan, Utah) and 1% penicillin/streptavidin (P/S) (PAN-Biotech, Aidenbach, Germany). DS-1 cells were maintained in RPMI-1640 supplemented with 10% FBS, 1% P/S and 1 ng/mL interleukin-6 (IL-6) (ThermoFisher Scientific). HEK-293T was maintained in DMEM (HyClone Laboratories) supplemented with 10% FBS and 1% P/S.

[0088] DS-1 and Nalm-6 were transduced with a murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retroviral vector (obtained from the St. Jude Children's Research Hospital Vector Development and Production Shared Resource, Memphis, Tenn.), containing the firefly luciferase gene and selected for GFP expression with a MoFlo cell sorter (Beckman Coulter, Brea, Calif.). DS-1 and OP-1 were transduced with a MSCV-IRES-GFP retroviral vector containing mCherry and selected for mCherry expression with a MoFlo cell sorter.

[0089] To induce differentiation of THP-1, 210.sup.6 THP-1 cells were cultured in 10 mL of RPMI-1640 supplemented with 10% FBS, 1% P/S, and 20 ng/ml phorbol 12-myristate 13-acetate (PMA) for 72 hours. The differentiated THP-1 cells were subsequently harvested using 1% EDTA (Merck, Kenilworth, N.J.).

[0090] Peripheral blood mononucleated cells were isolated by density gradient from discarded, anonymized by-products of platelet donations provided by the National University Hospital Blood Donation Centre. Mononucleated cells were cultured with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) in RPMI-1640 supplemented with 10% FBS, 1% P/S and 120 IU/ml interleukin-2 (IL-2) (Novartis, Basel, Switzerland) for 3 days. On day 4, anti-CD3/CD28 beads were removed and cells were restimulated with fresh anti-CD3/CD28 beads. Expanded T-cells were subsequently cultured in RPMI-1640 supplemented with 10% FBS, 1% P/S and 120 IU/ml IL-2.

Plasmids and Retroviral Transduction

[0091] The heavy chain and light chain domains of the anti-IL6 scFv in mb-aIL6 were derived from the published sequence of the human anti-IL6 monoclonal antibody AME-19a and joined with a 15 amino acid [(G4S).sub.3] linker to form a single chain variable fragment (scFv); the construct was synthesized by Genscript (Nanjing, China). The scFv was linked to the CD8 hinge and transmembrane domain (mb-aIL6). The anti-CD19-41BB-CD3 construct was previously developed in our laboratory..sup.12 The P2A sequence used to link mb-aIL6 and anti-CD19-41BB-CD3 was previously reported..sup.13 All constructs were subcloned into pMSCV-IRES-GFP between EcoRI and XhoI.

[0092] Preparation of retroviral supernatant and transduction were performed as previously described..sup.14 Briefly, T-cells were incubated with retroviral supernatant in the presence of RetroNectin (Takara, Kusatsu, Japan) at 37 C. Retroviral supernatant was replaced with freshly harvested supernatant every 12 hours thereafter for the next three days. Transduced T-cells were subsequently harvested and cultured in RPMI-1640 supplemented with 10% FBS, 1% P/S and 200 IU/mL IL-2.

Surface Staining of Transduced Cells

[0093] Surface expression of mb-aIL6 and anti-CD19-41BB-CD3c was detected by flow cytometry. For mb-aIL6, biotin-conjugated goat anti-human F(ab)2 (Jackson ImmunoResearch, West Grove, Pa.) was used followed by secondary staining with allophycocyanin (APC)-conjugated streptavidin (BD Biosciences, San Jose, Calif.). Cells were also labeled with human IL-6 conjugated to biotin (Abcam, Cambridge, UK), followed by streptavidin-APC; soybean-trypsin inhibitor conjugated to biotin (from R&D, Minneapolis, Minn.) was used as a negative control. CD19-myc, a soluble fusion protein produced by our laboratory containing the extracellular domain of human CD19 linked to a myc-tag, was used to specifically detect anti-CD19-41BB-CD3. T-cells were incubated with CD19-myc for 30 minutes, followed by R-phycoerythrin (PE)-conjugated anti-myc (Cell Signaling Technology, Danvers, Mass.). For cell immunophenotyping, T-cells were labeled with anti-CD3 APC, anti-CD56 PE, anti-CD4 V450, and anti-CD8 PerCP (BD Biosciences). Cell staining was analyzed using Accuri C6 or Fortessa flow cytometers (BD Biosciences).

IL-6 Depletion Assays

[0094] To measure IL-6 depletion, 210.sup.6 T cells were cultured in 1 mL RPMI-1640 containing 1 ng IL-6 for 2 hours. In another experiment, 0.5-210.sup.6 T cells were cultured in 1 mL RPMI-1640 containing 10 IU IL-6 for 2 hours. In yet another experiment, 210.sup.6 T cells were cultured in 1 mL RPMI-1640 containing 1 ng IL-6 for different time intervals of 20 minutes to 2 hours. In a further experiment, 0.510.sup.6 cells were cultured in 1 mL of RPMI containing 25-200 pg/mL recombinant human IL-6 for 2 hours at 37 C. In another further experiment, 0.210.sup.6 T cells were cultured in 1 mL RPMI-1640 containing 1 ng IL-6 for 2-72 hours. At the end of the cultures, the supernatant was harvested, filtered with a 0.22 m filter, and diluted at 1:10. Levels of IL-6 were measured by ELISA using the Human IL-6 Platinum ELISA kit (ThermoFisher). Interpolation from the calculated standard curve was used to determine the concentration of IL-6 in each sample.

[0095] For Stat3 phosphorylation measurements, 210.sup.6 cells were seeded in 1 mL RPMI-1640 containing 10 IU IL-6 for 2 hours. Supernatant was harvested and incubated with 0.210.sup.6 U937 cells for 15 minutes at 37 C. Lysefix buffer (BD Bioscience) was added and samples were further kept for 10 minutes at 37 C. The cells were then washed and placed in Perm III Buffer for 30 minutes on ice. After three washes, PE-conjugated anti-Stat3 (pY705) antibody (BD Biosciences) was added. After 1 hour, cells were washed and analyzed by flow cytometry.

[0096] To determine the effect of IL-6 depletion by mb-aIL6 on the growth of the IL-6-dependent cell line DS-1, 1.510.sup.4 T cells were incubated with DS-1 cells transduced with mCherry at 1:1 ratio in RPMI-1640 supplemented with 10% FBS, 1% P/S, and 0.5 ng/ml IL-6. For expanded T-cells, 120 IU/ml IL-2 was added. DS-1 cells had been starved for 72 hours before initiation of the experiments. IL-2 and IL-6 were added every 2 days during the culture. A 4 image of each well was captured every 4 hours using the IncuCyte Live Cell Analysis System (Essen Biosciences, Ann Arbor, Mich.). Cell count was measured using fluorescence and the total red object integrated intensity was used as a measure for the amount of DS-1 mCherry in the well. In other experiments, similar culture were performed with T cells, OP-1 and differentiated THP-1 at a 1:5:1 ratio. Differentiated THP-1 were seeded 1 hour before the beginning of the assay. A 4 image of each well was captured as above using IncuCyte system. After 40 hours, the supernatant from each well was harvested, diluted at 1:5, and IL-6 measured by ELISA as described above.

Interferon- (IFN) Production, CD107a Expression, Cytotoxicity and Proliferation Assays

[0097] To test for IFN production, 110.sup.5 T cells were cultured with OP-1 cells at a 1:1 ratio. After 1 hour, GolgiPlug (BD Biosciences) was added to the cells, which were cultured for another 5 hours. After cell membrane permeabilization with 8E, a permeabilization reagent developed in our laboratory, cells were labelled with PE-conjugated anti-human IFN antibody (BD Biosciences), and analyzed by flow cytometry.

[0098] To measure exocytosis of cytotoxic granules, cells were cultured as described above. At the beginning of the cultures, PE-conjugated anti-human CD107a antibody (BD Biosciences) was added. After 1 hour, GolgiStop (BD Biosciences) was added and the cultures continued for another 3 hours before analysis by flow cytometry.

[0099] To test cytotoxicity, OP-1 cells were labelled with calcein-AM Red (Invitrogen, Carlsbad, Calif.) and plated into a 96-well round-bottom plate. T cells (110.sup.5) were added at a 1:1 E:T ratio and co-cultured for 4 hours. Viable target cells (Calcein-AM positive) were counted by flow cytometry, as previously described..sup.14

[0100] To measure cell proliferation, 110.sup.5 T cells were co-cultured with irradiated OP-1 at 1:1 E:T ratio in RPMI-1640 supplemented with 10% FBS, 1% P/S and 120 IU/ml IL-2. IL-2 was added every 2 days into each well. On day 7, 14, and 21, T cells were counted by flow cytometry. Fresh irradiated OP-1 cells were added after cell counting to reconstitute the 1:1 E:T ratio.

Xenograft Experiments

[0101] DS-1 cells expressing luciferase were injected intraperitoneally (i.p.; 110.sup.6 cells/mouse) in NOD.Cg-Prkdc.sup.scid IL2rg.sup.tm1Wj1/SzJ (NOD/scid IL2RGnull) mice (Jackson Laboratory). Two days after DS-1 inoculation, mice received an i.p. injection of either 110.sup.7 T cells transduced with GFP alone, 110.sup.7 T cells transduced with MSCV-mb-aIL6, or RPMI 1640 with 10% FBS instead of T cells. All mice received 20 000 IU of IL-2 and 1000 IU of IL-6 i.p. every 2 days. Tumor load was determined twice a week using the Xenogen IVIS-200 System (Caliper Life Sciences, Waltham, Mass.) after injecting aqueous d-luciferin potassium salt (Perkin Elmer, Waltham, Mass.) i.p. (2 mg per mouse). Luminescence was measured with the Living Image 3.0 software.

[0102] Nalm-6 cells expressing luciferase were injected intravenously (i.v.; 0.510.sup.6 cells/mouse for FIGS. 10A-B, 110.sup.6 cells/mouse for FIGS. 10C-F) in NOD/scid IL2RGnull mice. Three days later, mice received an i.v. injection of either 210.sup.7 T cells transduced with the anti-CD19-41BB-CD3 CAR, 210.sup.7 T cells transduced with the construct containing the CAR and mb-aIL6 (DUAL), or RPMI 1640 with 10% FBS instead of T cells. All mice received 20 000 IU of IL-2 intraperitoneally every 2 days. Tumor load was determined twice a week using the Xenogen IVIS-200 System (Caliper Life Sciences, Waltham, Mass.) after injecting aqueous d-luciferin potassium salt (Perkin Elmer, Waltham, Mass.) i.p. (2 mg per mouse). Luminescence was analyzed with the Living Image 3.0 software.

Results

Design, Expression, and Specificity of Mb-aIL6

[0103] To generate a membrane-bound anti-IL6 construct, a single chain variable fragment (scFv) was synthesized from the sequences of the variable light and heavy chains of the human anti-IL-6 antibody AME-19a and linked to the hinge and transmembrane domains of CD8 (FIG. 1A). The construct was placed in an MSCV retroviral vector containing IRES and GFP (FIG. 1B). This retroviral vector was used to transduce Jurkat T cells. GFP expression was high in cells transduced with the MSCV-mb-aIL6: 98% of cells were GFP-positive. To detect mb-aIL6 on the surface of the transduced Jurkat cells, the cells were labeled with a biotin-conjugated goat anti-human F(ab) antibody followed by streptavidin-APC. As shown in FIG. 1C, mb-aIL6 was detected in essentially all GFP-expressing Jurkat cells, whereas cells transduced with a vector containing GFP alone (Mock) were mb-aIL6-negative.

[0104] To determine whether mb-aIL6 expressed on the cell surface could bind human IL-6, the transduced Jurkat cells were exposed to biotin-conjugated human IL-6 for 10 minutes; cells were then labelled with streptavidin-APC. As shown in FIG. 1D, mb-aIL6-Jurkat cells bound IL-6 at levels that were proportional to levels of GFP and, hence, receptor expression: 99% of cells bound IL-6, whereas cells labelled with a biotinylated control protein (soybean-trypsin inhibitor) remained unstained.

Neutralization of IL-6 with Mb-aIL6 Cells

[0105] Binding of mb-aIL6 to IL-6 was corroborated by experiments in which Jurkat cells were cultured for 2 hours in medium containing recombinant human IL-6 (1 ng/mL); residual IL-6 recovered after culture was measured in the supernatant by ELISA. After 2 hours of culture, concentration of IL-6 in the supernatant from the mb-aIL6 Jurkat cells was 0.163 ng/mL versus 0.941 ng/mL in the supernatant of mock-transduced Jurkat cells (FIG. 2A).

[0106] To determine the number of mb-aIL6 Jurkat cells required to neutralize IL-6, increasing concentrations of cells from 0.2510.sup.6 cells/mL to 210.sup.6 cells/mL were used. Removal of IL-6 from the supernatant was cell-dose dependent (FIG. 2B). In parallel experiments, the kinetics of IL-6 removal by mb-aIL6 cells was measured. As shown in FIG. 2C, IL-6 neutralization was time-dependent, with nearly 90% being neutralized after 30 minutes, and becoming undetectable after 120 minutes. Notably, the curve fits that of a first order exponential decay curve (R.sup.2=0.9957), indicating an IL-6 half-life of 7.443 minutes and a K of 0.09313.

[0107] To test whether mb-aIL6 expressing Jurkat cells could also neutralize low concentrations of IL-6, an IL-6 depletion assay was set up using concentrations of IL-6 ranging from 0.025 to 0.2 ng/mL. As shown in FIG. 2D, mb-aIL6 expressing Jurkat cells could neutralize most IL-6 just as well. Consistent with previous experiments, there was a 3.8-5.4 fold decrease in levels of IL-6 across the various concentrations by mb-aIL6 expressing Jurkat cells as compared to mock-transduced cells.

[0108] We postulated that cell proliferation would generate new mb-aIL6 cells which would continue to neutralize IL-6 in prolonged cell cultures. To test this notion, we used culture conditions previously determined (FIG. 2B) that were insufficient to neutralize IL-6 in 2 hours, and continue the cultures for 72 hours. At 24 hour, most of the IL-6 had been removed from the supernatant (FIG. 2E).

Functional Consequences of IL-6 Neutralization with Mb-aIL6 T Cells

[0109] U937 is a monocyte cell line that can be stimulated by IL-6..sup.15,16 Upon binding to the IL-6 receptor, IL-6 triggers Stat3 phosphorylation..sup.17 The capacity of IL-6-containing supernatant, after exposure to mb-aIL6- or mock-transduced Jurkat cells for 2 hours, was tested to trigger Stat3 phosphorylation in U937. After 15 minutes of exposure to the supernatant containing 1 ng/mL IL-6, Stat3 phosphorylation was readily detected (P<0.001; n=3; FIG. 3A). A similar levels of phosphorylation was observed if the IL-6 supernatant had been collected from cultures with mock-transduced Jurkat cells (P=not significant). By contrast, when the cultures contained mb-aIL6 Jurkat cells, levels of Stat3 phosphorylation were markedly lower than those measured in U937 cells exposed to IL-6 or to IL-6-containing supernatant of mock-transduced Jurkat cells (P<0.001 for either comparison), and similar to those of unstimulated cells (P=not significant).

[0110] DS-1 is a B-lymphoma cell line whose proliferation requires IL-6..sup.18 We determined whether co-culture with Jurkat cells expressing mb-aIL6 would affect DS-1 expansion. For this purpose, sequential live cell imaging recording of mCherry-transduced DS-1 cells over 5 days was used. As shown in FIG. 3B, DS-1 cultured in IL-6 containing medium, in the presence of mock-transduced Jurkat rapidly expanded whereas expansion was markedly reduced if mb-aIL6-transduced Jurkat cells were present in the cultures. Under these conditions, DS-1 growth rate was similar to that observed in cultures lacking IL-6, regardless of whether mock- or mb-aIL6-transduced Jurkat cells were present. Collectively, these results corroborate the capacity of cells expressing mb-aIL6 to neutralize the effects of IL-6.

Expression of Mb-aIL6 in Human Peripheral Blood T-Lymphocytes

[0111] In the next set of experiments, whether mb-aIL-6 could be expressed on the surface of peripheral blood T lymphocytes was determined. For this purpose, peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 beads and then transduced with the MSCV-mb-aIL6 retroviral vector. Cells transduced with a vector containing GFP alone (Mock) were used as controls. As shown in FIGS. 4A and 4B, mb-aIL6 was highly expressed on the surface of transduced GFP+T lymphocytes, and effectively bound IL-6. The immunophenotype of T cells expressing mb-IL6 remained essentially identical to that of mock-transduced T cells (FIG. 4C).

[0112] Whether T cells expressing mb-IL6 could suppress the growth of the IL-6-dependent cell line DS-1 was tested. FIG. 4D shows that DS-1 growth was markedly suppressed, indicating that mb-aIL6 expressed in T lymphocytes could neutralize IL-6 as well as Jurkat cells expressing the receptor.

Membrane-Bound Anti-IL-6 and Anti CD19-41BB-CD3z CAR can be Co-Expressed in T Lymphocytes

[0113] If CAR T cells were to express mb-aIL6 in addition to CARs, this might prevent the occurrence of CRS by neutralizing IL-6 secreted by activated T lymphocytes and by macrophages in the microenvironment.

[0114] As a first step to test this notion, whether mb-aIL6 and CAR could be effectively expressed simultaneously was determined. To this end, a bicistronic MSCV vector containing genes encoding mb-aIL6 and an anti-CD19-41BB-CD3 CAR (FIG. 5A) was developed..sup.12 To specifically detect the anti-CD19 CAR, the extracellular domain of the human CD19 molecule was linked to a myc tag and cells were stained with an anti-myc antibody. FIG. 5B shows that both mb-aIL6 and the anti-CD19 CAR could be expressed at high levels in peripheral blood T cells. As with mb-aIL6 expressed alone, mb-aIL6 expressed in conjunction with the CAR effectively neutralized IL-6 (FIG. 5C).

Expression of Mb-aIL6 does not Affect CAR T-Cell Function

[0115] Whether expression of mb-aIL6 and IL-6 neutralization would affect the T cell functions activated by the CAR was determined. Using T lymphocytes from 3 donors expressing either mb-aIL6 or anti-CD19 CAR and T cells expressing both receptors, production of IFN after co-culture with CD19+ target cells OP-1 was tested. Production of IFN in cells expressing the CAR was high, regardless of whether mb-aIL6 was expressed, whereas T cells expressing mb-aIL6 alone secreted levels of IFN similar to those of mock-transduced cells (FIG. 6A).

[0116] In the presence of CD19+ target cells, CAR-expressing cells released cytotoxic granules as evidenced by staining with the anti-CD107a antibody. Percentage of CD107a+ cells was similar, regardless of mb-aIL6 expression, while cells expressing mb-aIL6 alone remained CD107a-negative (FIG. 6B). In line with this result, percent cytotoxicity against CD19+ targets driven by the CAR remains unchanged by the presence of mb-aIL6 (FIG. 6C).

[0117] An important functional property of second- and later-generation CARs is the capacity to provide co-stimulation and sustain prolonged T cell proliferation..sup.19 As shown in FIG. 6D, anti-CD19 CAR-T cells proliferated for 4 weeks in the presence of CD19+ target cells, and the rate of proliferation was similar in cells with and without mb-aIL6. By contrast, cells transduced with mb-aIL6 only and mock-transduced cells did not expand, and expansion did not occur in the absence of target cells regardless of CAR expression. Together, these results indicate that expression of mb-aIL6 does not affect CAR-driven T cell secretion of IFN, specific cytotoxicity or cell proliferation.

T Cells Expressing Mb-aIL6 and CAR can Kill Target Cells while Neutralizing IL-6

[0118] During CRS triggered by CAR activation, IL-6 secreted by macrophages contributes to its severity..sup.10

[0119] To mimic the interaction between CAR-T cells and macrophages, CAR-T cells were co-cultured with the monocyte cell line THP-1, which secretes IL-6 in the presence of TNF- and IFN;.sup.20 the latter cytokines, together with IL-6, are secreted by T-lymphocytes upon activation..sup.9,21 Thus, T-lymphocytes transduced with mb-aIL6 and/or anti-CD19 CAR, THP-1, and CD19+ leukemic cells OP-1 were co-cultured for 40 hours (FIG. 7A). Killing of OP-1 cells and levels of IL-6 in the supernatant were monitored. As shown in FIG. 7B, CAR T cells effectively kill OP-1 regardless of mb-aIL6 expression or presence of THP-1. Remarkably, levels of IL-6 were considerably elevated in cultures containing both CAR-T and THP-1 cells but these were essentially undetectable if the CAR-T cells also expressed mb-aIL6.

Xenograft Experiments

[0120] To determine the capacity of mb-aIL6 expressing T lymphocytes to neutralize IL-6 in vivo, experiments with NOD/scid IL2RGnull mice engrafted with luciferase-labeled DS-1 cells were performed. Tumor growth was measured by live imaging, and compared mice that received T cells transduced with GFP alone and mice that received T cells transduced with mb-aIL6. In two of three mice that received mb-aIL6 transduced T cells, there was a reduction in tumor burden, while tumor burden in mice that received mock-transduced T cells remained stable or increased (FIGS. 9A-B).

[0121] Whether expression of mb-aIL6 affected the antitumor capacity of anti-CD19 CAR T cells was tested. Experiments were performed with NOD/scid IL2RGnull mice engrafted with luciferase-labeled Nalm-6 cells. Tumor growth was measured by live imaging and compared mice that received T cells transduced with CAR alone and mice that received T cells transduced with the bicistronic construct containing CAR and mb-aIL6. CAR T cells exerted anti-leukemic activity regardless of whether mb-aIL6 was expressed (FIGS. 10A-F). Together, these results suggest that mb-aIL6 can neutralize IL-6 in vivo, and does not significantly affect CAR function.

Discussion

[0122] The results of this study indicate that mb-aIL6 carried by T lymphocytes is a powerful neutralizer of IL-6. Importantly, mb-aIL6 can be expressed on T lymphocytes in combination with CARs without affecting CAR potency. To this end, CAR-T cells expressing mb-aIL6 should have less risk of triggering severe CRS, while retaining their anti-tumor potential.

[0123] We have now designed other receptors which are functionally related to mb-aIL6 but have distinctive features (FIGS. 8A-C). FIG. 8A is a schematic of a nucleic acid construct for a soluble form of the receptor lacking the CD8 transmembrane domain of mb-aIL6 (sec-aIL6), which would be continuously secreted by transduced cells, and might diffuse to areas of the inflammatory microenvironment more effectively. FIG. 8B is a schematic of a nucleic acid construct where mb-aIL6 is linked to stimulatory and co-stimulatory domains typically incorporated in CARs (aIL6-CAR). Cells bearing such receptor should proliferate upon ligation of the aIL6-CAR, thus magnifying IL-6 neutralization. FIG. 8C is a schematic of a nucleic acid construct where the scFv anti-IL6 is replaced by the IL-6 receptor deprived of signaling capacity. This is constituted by the extracellular domain of gp130 fused to the IL-6 receptor and the hinge plus transmembrane domain of CD8. The potential advantage of this format is that it may be less immunogenic that the scFv-containing receptor.

[0124] While this study focused on IL-6, a similar approach can be applied to neutralize other pro-inflammatory cytokines, such as tumor necrosis factor (TNF)- (FIGS. 11A-B), IL-1, IL-12, IL-17, IL-18, IFN, etc., and/or block their receptors. For example, based on the schema in FIG. 11A, the anti-IL6 scFv portion can be replaced with a different scFv that specifically bind to a different cytokine, such as (TNF)- (FIG. 11), IL-1, IL-12, IL-17, IL-18, or IFN. Multiple neutralizing receptors can be expressed on the same cells or in different cell subsets to exert a comprehensive and long-lasting anti-inflammatory effect.

TABLE-US-00001 SEQUENCES mb-aIL6Sequence CD8signalpeptidenucleotidesequence(SEQIDNO:1): ATGGCCCTGCCCGTGACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTGCATGCTGC TAGACCC CD8signalpeptideaminoacidsequence(SEQIDNO:2): MALPVTALLLPLALLLHAARP Variablelightchainnucleotidesequenceofanti-IL6(SEQIDNO:3): GAAATCGTCCTGACCCAGTCCCCTGCCACACTGTCCCTGTCTCCAGGAGAGAGGG CCACCCTGAGCTGCTCCGCCTCTATCAGCGTGTCCTACATGTATTGGTACCAGCA GAAGCCAGGACAGGCACCTAGGCTGCTGATCTACGACATGTCTAACCTGGCAAG CGGCATCCCCGCACGCTTCTCTGGAAGCGGATCCGGCACAGACTTTACACTGACC ATCAGCTCCCTGGAGCCTGAGGATTTCGCCGTGTACTATTGCATGCAGTGGTCCG GCTATCCATACACATTTGGCGGCGGCACCAAGGTGGAGATCAAG VariablelightchainaminoacidsequenceofantiIL-6(SEQIDNO:4): EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLASGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIK GSGlinkernucleotidesequence(SEQIDNO:5): GGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCC GSGlinkeraminoacidsequence(SEQIDNO:6): GGGGSGGGGSGGGGS VariableheavychainnucleotidesequenceofantiIL-6(SEQIDNO:7): GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTG CGGCTGTCTTGTGCCGCCAGCGGCTTCACCTTTTCTCCATTCGCCATGAGCTGGGT GAGACAGGCACCAGGCAAGGGCCTGGAGTGGGTGGCCAAGATCTCCCCTGGCGG CTCTTGGACATACTATTCCGACACAGTGACCGGCCGGTTTACCATCTCCAGAGAT AACGCCAAGAACAGCCTGTATCTGCAGATGAATAGCCTGCGGGCCGAGGACACA GCCGTGTACTATTGTGCCAGACAGCTGTGGGGCTACTATGCCCTGGATATCTGGG GCCAGGGCACCACAGTGACCGTGTCTAGC VariableheavychainaminoacidsequenceofantiIL-6(SEQIDNO:8): EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAKISPGGS WTYYSDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYALDIWGQ GTTVTVSS CD8hingeandtransmembranedomainnucleotidesequence(SEQIDNO:9): AAGCCTACCACAACCCCAGCACCCAGGCCCCCTACACCTGCACCAACCATCGCC AGCCAGCCACTGTCCCTGAGGCCCGAGGCATGCAGGCCTGCAGCAGGAGGCGCC GTGCACACCCGCGGCCTGGACTTCGCCTGTGATATCTACATCTGGGCACCCCTGG CTGGAACCTGCGGAGTCCTGCTGCTGTCACTGGTCATTACCCTGTATTGC CD8hingeandtransmembranedomainaminoacidsequence(SEQIDNO:10): KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYC sec-aIL6 sec-aIL6nucleotidesequence(SEQIDNO:11): ATGGCCCTGCCCGTGACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTGCATGCTGC TAGACCCGAAATCGTCCTGACCCAGTCCCCTGCCACACTGTCCCTGTCTCCAGGA GAGAGGGCCACCCTGAGCTGCTCCGCCTCTATCAGCGTGTCCTACATGTATTGGT ACCAGCAGAAGCCAGGACAGGCACCTAGGCTGCTGATCTACGACATGTCTAACC TGGCAAGCGGCATCCCCGCACGCTTCTCTGGAAGCGGATCCGGCACAGACTTTAC ACTGACCATCAGCTCCCTGGAGCCTGAGGATTTCGCCGTGTACTATTGCATGCAG TGGTCCGGCTATCCATACACATTTGGCGGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGAGGTGCAGCT GGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGCGGCTGTCTTG TGCCGCCAGCGGCTTCACCTTTTCTCCATTCGCCATGAGCTGGGTGAGACAGGCA CCAGGCAAGGGCCTGGAGTGGGTGGCCAAGATCTCCCCTGGCGGCTCTTGGACA TACTATTCCGACACAGTGACCGGCCGGTTTACCATCTCCAGAGATAACGCCAAGA ACAGCCTGTATCTGCAGATGAATAGCCTGCGGGCCGAGGACACAGCCGTGTACT ATTGTGCCAGACAGCTGTGGGGCTACTATGCCCTGGATATCTGGGGCCAGGGCAC CACAGTGACCGTGTCTAGC sec-aIL6aminoacidsequence(SEQIDNO:12): MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQ KPGQAPRLLIYDMSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPY TFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSP FAMSWVRQAPGKGLEWVAKISPGGSWTYYSDTVTGRFTISRDNAKNSLYLQMNSLR AEDTAVYYCARQLWGYYALDIWGQGTTVTVSS aIL6BBzSequence CD8signalpeptidenucleotidesequence(SEQIDNO:13): ATGGCCCTGCCCGTGACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTGCATGCTGC TAGACCC CD8signalpeptideaminoacidsequence(SEQIDNO:14): MALPVTALLLPLALLLHAARP aIL6scFvnucleotidesequence(SEQIDNO:15): GAAATCGTCCTGACCCAGTCCCCTGCCACACTGTCCCTGTCTCCAGGAGAGAGGG CCACCCTGAGCTGCTCCGCCTCTATCAGCGTGTCCTACATGTATTGGTACCAGCA GAAGCCAGGACAGGCACCTAGGCTGCTGATCTACGACATGTCTAACCTGGCAAG CGGCATCCCCGCACGCTTCTCTGGAAGCGGATCCGGCACAGACTTTACACTGACC ATCAGCTCCCTGGAGCCTGAGGATTTCGCCGTGTACTATTGCATGCAGTGGTCCG GCTATCCATACACATTTGGCGGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCG GCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGAGGTGCAGCTGGTGGAG AGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGCGGCTGTCTTGTGCCGCCA GCGGCTTCACCTTTTCTCCATTCGCCATGAGCTGGGTGAGACAGGCACCAGGCAA GGGCCTGGAGTGGGTGGCCAAGATCTCCCCTGGCGGCTCTTGGACATACTATTCC GACACAGTGACCGGCCGGTTTACCATCTCCAGAGATAACGCCAAGAACAGCCTG TATCTGCAGATGAATAGCCTGCGGGCCGAGGACACAGCCGTGTACTATTGTGCCA GACAGCTGTGGGGCTACTATGCCCTGGATATCTGGGGCCAGGGCACCACAGTGA CCGTGTCTAGC aIL6scFvaminoacidsequence(SEQIDNO:16): EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLASGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIKGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAKI SPGGSWTYYSDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYAL DIWGQGTTVTVSS CD8hingeandtransmembranedomainnucleotidesequence(SEQIDNO:17): ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGA CTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC CD8hingeandtransmembranedomainaminoacidsequence(SEQIDNO:18): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV LLLSLVITLYC 41BBdomainnucleotidesequence(SEQIDNO:19): AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG 41BBdomainaminoacidsequence(SEQIDNO:20): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3domainnucleotidesequence(SEQIDNO:21): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC CCTGCCCCCTCGC CD3domainaminoacidsequence(SEQIDNO:22): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR mb-gp130-IL6RSequence gp130domainnucleotidesequence(SEQIDNO:23): ATGCTGACACTGCAGACATGGCTGGTCCAGGCACTGTTTATCTTTCTGACAACCG AGTCCACAGGCGAACTGCTGGATCCTTGCGGGTACATCTCTCCAGAGAGCCCCGT GGTGCAGCTGCACTCCAACTTCACCGCCGTGTGCGTGCTGAAGGAGAAGTGTATG GACTACTTTCACGTGAACGCCAATTATATCGTGTGGAAGACAAACCACTTCACCA TCCCTAAGGAGCAGTACACAATCATCAATAGAACCGCCAGCTCCGTGACCTTCAC CGATATCGCCAGCCTGAACATCCAGCTGACATGCAATATCCTGACCTTCGGCCAG CTGGAGCAGAACGTGTATGGCATCACCATCATCTCCGGCCTGCCCCCTGAGAAGC CAAAGAACCTGTCTTGCATCGTGAATGAGGGCAAGAAGATGAGGTGTGAGTGGG ACCGGGGCAGAGAGACACACCTGGAGACAAATTTCACCCTGAAGTCCGAGTGGG CCACCCACAAGTTTGCCGACTGCAAGGCCAAGAGGGATACACCCACCAGCTGTA CAGTGGATTACTCCACCGTGTATTTTGTGAACATCGAAGTGTGGGTGGAGGCCGA GAATGCCCTGGGCAAGGTGACCAGCGACCACATCAACTTCGATCCCGTGTACAA GGTGAAGCCTAACCCACCCCACAATCTGTCTGTGATCAATAGCGAGGAGCTGTCT AGCATCCTGAAGCTGACATGGACCAACCCCTCCATCAAGTCTGTGATCATCCTGA AGTACAATATCCAGTATAGAACAAAGGACGCCAGCACCTGGTCCCAGATCCCTC CAGAGGATACAGCCTCCACCAGGTCCTCTTTTACAGTGCAGGACCTGAAGCCTTT CACCGAGTACGTGTTCCGGATCCGGTGTATGAAGGAGGACGGCAAGGGCTACTG GTCTGATTGGAGCGAGGAGGCCTCCGGCATCACCTATGAGGACAGGCCA gp130domainaminoacidsequence(SEQIDNO:24): MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYF HVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGI TIISGLPPEKPKNLSCIVNEGKKMRCEWDRGRETHLETNFTLKSEWATHKFADCKAK RDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVIN SEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFT EYVFRIRCMKEDGKGYWSDWSEEASGITYEDRP GSGlinkernucleotidesequence(SEQIDNO:25): GGAGGAGGAGGAAGCGGAGGAGGAGGCTCCGGCGGCGGCGGCTCT GSGlinkeraminoacidsequence(SEQIDNO:26): GGGGSGGGGSGGGGS IL-6Rdomainnucleotidesequence(SEQIDNO:27): GTGGATGTGCCCCCTGAGGAGCCCCAGCTGTCTTGCTTCAGGAAGTCCCCTCTGT CTAACGTGGTGTGCGAGTGGGGACCTCGCAGCACCCCATCCCTGACCACAAAGG CCGTGCTGCTGGTGCGGAAGTTCCAGAATAGCCCTGCCGAGGACTTTCAGGAGCC ATGCCAGTACTCTCAGGAGAGCCAGAAGTTCAGCTGTCAGCTGGCAGTGCCAGA GGGCGATAGCTCCTTTTATATCGTGTCCATGTGCGTGGCCTCTAGCGTGGGCTCC AAGTTCTCTAAGACACAGACCTTTCAGGGCTGTGGCATCCTGCAGCCTGACCCAC CCGCCAACATCACAGTGACCGCCGTGGCCCGGAATCCAAGATGGCTGTCTGTGA CATGGCAGGATCCCCACAGCTGGAACTCCTCTTTCTACCGGCTGAGATTTGAGCT GAGGTATCGCGCCGAGCGGAGCAAGACCTTTACCACATGGATGGTGAAGGACCT GCAGCACCACTGCGTGATCCACGATGCATGGAGCGGCCTGAGGCACGTGGTGCA GCTGAGAGCACAGGAGGAGTTCGGACAGGGAGAGTGGAGCGAGTGGTCCCCAG AGGCAATGGGAACACCATGGACCGAGAGCCGCTCCCCTCCAGCAGAGAATGAGG TGAGCACACCA IL-6Rdomainaminoacidsequence(SEQIDNO:28): VDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQY SQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTA VARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHD AWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTP CD8hingeandtransmembranedomainnucleotidesequence(SEQIDNO:29): AAGCCAACCACAACCCCTGCACCACGGCCCCCTACACCAGCACCTACCATCGCAT CCCAGCCACTGTCTCTGAGGCCTGAGGCATGCAGGCCAGCAGCAGGAGGAGCAG TGCACACCCGGGGCCTGGACTTCGCCTGTGATATCTACATCTGGGCCCCACTGGC TGGCACTTGCGGGGTCCTGCTGCTGTCCCTGGTCATCACTCTGTATTGC CD8hingeandtransmembranedomainaminoacidsequence(SEQIDNO:30): KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYC mb-aTNFSequence CD8signalpeptidenucleotidesequence(SEQIDNO:31): ATGGCCCTGCCTGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCCG CCCGCCCC CD8signalpeptideaminoacidsequence(SEQIDNO:32): MALPVTALLLPLALLLHAARP VariablelightchainnucleotidesequenceofantiTNF(SEQIDNO:33): GAAATCGTCCTGACCCAGTCCCCCGCCACACTGTCTCTGAGCCCAGGAGAGAGG GCCACCCTGAGCTGCAGAGCCTCCCAGTCTGTGAGCTCCTACCTGGCCTGGTATC AGCAGAAGCCAGGACAGGCACCAAGGCTGCTGATCTACGACGCATCCAACAGGG CAACAGGCATCCCCGCACGCTTCAGCGGATCCGGATCTGGCAGCGGCACCGACT TTACACTGACCATCTCTAGCCTGGAGCCTGAGGATTTCGCCGTGTACTATTGCCA GCAGCGCAGCAATTGGCCCCCTTTCACATTTGGCCCAGGCACCAAGGTGGATATC AAG VariablelightchainaminoacidsequenceofantiTNF(SEQIDNO:34): EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP ARFSGSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIK GSGlinkernucleotidesequence(SEQIDNO:35): GGAGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCAGC GSGlinkeraminoacidsequence(SEQIDNO:36): GGGGSGGGGSGGGGS VariableheavychainnucleotidesequenceofantiTNF(SEQIDNO:37): CAGGTGCAGCTGGTGGAGTCCGGCGGCGGCGTGGTGCAGCCAGGCAGGTCCCTG AGGCTGTCTTGTGCAGCAAGCGGCTTCATCTTTTCCTCTTACGCAATGCACTGGGT GCGGCAGGCACCTGGAAACGGCCTGGAGTGGGTGGCCTTCATGTCCTACGACGG CTCTAATAAGAAGTATGCCGATTCCGTGAAGGGCCGGTTTACAATCAGCAGAGA CAACTCCAAGAATACCCTGTATCTGCAGATGAACTCTCTGAGGGCCGAGGACAC AGCCGTGTACTATTGTGCCCGGGATAGAGGAATCGCAGCAGGAGGAAATTACTA TTACTATGGCATGGACGTGTGGGGCCAGGGCACCACAGTGACCGTGAGCTCC VariableheavychainaminoacidsequenceofantiTNF(SEQIDNO:38): QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDG SNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYY YGMDVWGQGTTVTVSS CD8hingeandtransmembranedomainnucleotidesequence(SEQIDNO:39): AAGCCTACCACAACCCCTGCACCACGGCCACCAACACCAGCACCTACCATCGCCT CTCAGCCTCTGAGCCTGAGGCCAGAGGCATGCAGGCCAGCAGCAGGAGGAGCAG TGCACACCAGAGGCCTGGACTTTGCCTGTGATATCTACATCTGGGCCCCTCTGGC TGGGACTTGCGGGGTGCTGCTGCTGTCACTGGTCATCACACTGTATTGTTGA CD8hingeandtransmembranedomainaminoacidsequence(SEQIDNO:40): KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYC

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INCORPORATION BY REFERENCE; EQUIVALENTS

[0146] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0147] While example embodiments have been particularly shown and described, 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.