Hybrid Anti-CD20 car t cell therapy for the treatment of autoimmune diseases

Abstract

The present disclosure provides a method of treating autoimmune diseases such as multiple sclerosis, comprising the use of anti-CD20 chimeric antigen receptor (CAR) that contains a hybrid single chain variable fragment (scFv), wherein the framework regions (FRs) and complementarity-determining regions (CDRs) of the scFv are derived from different anti-CD20 antigen binding regions or anti-CD20 antibodies. Furthermore, the CAR comprises a torsional linker (e.g. 1-4 alanine residues) between the transmembrane domain and the cytoplasmic region of the CAR.

Description

HYBRID ANTI-CD20 CAR T CELL THERAPY FOR THE TREATMENT OF AUTOIMMUNE DISEASESSEQUENCE LISTING STATEMENT

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 28, 2025, is named P-639689-PCT_10_28_2025_SL.xml and is 35,389bytes in size.FIELD OF THE INVENTION

[0002] This invention relates generally to the field of immunotherapy. In one embodiment, the present disclosure provides a method of treating autoimmune diseases.BACKGROUND OF THE INVENTION

[0003] Autoimmune diseases are conditions in which our immune system mistakenly damages healthy cells in our body. There are over 100 known autoimmune diseases. Common ones include type 1 diabetes, rheumatoid arthritis, psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison’s disease, Graves’ disease, Sjogren's disease, Hashimoto’s thyroiditis, myasthenia gravis, celiac disease, autoimmune vasculitis, and pernicious anemia.

[0004] Traditionally, autoimmune disorders were classified as T cell mediated or autoantibody mediated. However, improved understanding of the complexity of the immune system has significantly influenced the way we view autoimmune diseases and their pathogeneses. Reciprocal roles of T-cell help for B cells during adaptive immune responses and B-cell help in CD4+ T-cell activation are being increasingly recognized. The observation that most autoantibodies in traditionally autoantibody-mediated diseases are of the IgG isotype and carry somatic mutations strongly suggests T-cell help in the autoimmune B-cell response. Likewise, B cells function as crucial antigen presenting cells in autoimmune diseases that are traditionally viewed as T cell mediated.

[0005] Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS). It affects approximately 1 million patients in the United States and is the most common cause of irreversible neurologic disability in young adults. The clinical course of MS includes neurological deficits, weakness, vision loss and cognitive decline. MS is categorized in 3 variants comprising (i) relapsing-remitting MS (RRMS), representing 80-90% of all cases, (ii) secondary progressive MS (SPMS) which follows 70-80% of RRMS cases 10-15 years after disease onset and (iii) primary progressive MS representing 10-15% of all cases. There is a need to develop improved therapy for MS that targets the CNS.SUMMARY OF THE INVENTION

[0006] In one embodiment, provided herein is use of a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) to treat an autoimmune disease. In one embodiment, provided herein is a method of treating an autoimmune disease in a patient, comprising administering to the patient a composition comprising an effecti ve amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR). In some embodiments, the anti-CD20 CAR comprises a hybrid anti-CD20 single chain variable fragment (scFv) comprising framework regions (FRs) and complementarity-determining regions (CDRs) that are derived from (i) different antigen binding regions or antibodies that bind CD20, or (ii) anti-CD20 antigen binding regions or anti-CD20 antibodies of different tonic signaling intensities. In some embodiments, the hybrid anti-CD20 scFv comprises (i) a light chain variable region (VL) comprising in order from amino-proximal to carboxy-proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); and (ii) a heavy chain variable region (VH) comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity-determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity-determining region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26,27, 18, 8, 9, and 10, respectively, or SEQ ID NOs: 15, 16, 17, 18, 20, 9 and 21 respectively, and wherein the FIFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs: 11, 12, 13, 14, 5, 6, and 19, respectively. In some embodiments, the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, and the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7 respectively. In some embodiments, the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 15, 16, 17, 18, 20, 9 and 21, respectively, and the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs: 11, 12, 13, 14, 5, 6, and 19 respectively.

[0007] In one embodiment, provided herein is use of a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) to treat multiple sclerosis. In one embodiment, provided herein is a method of treating multiple sclerosis in a patient, comprising administering to the patient a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR). In some embodiments, the anti-CD20 CAR comprises a hybrid anti-CD20 single chain variable fragment (scFv) comprising framework regions (FRs) and complementarity-determining regions (CDRs) that are derived from (i) different antigen binding regions or antibodies that bind CD20, or (ii) anti-CD20 antigen binding regions or anti-CD20 antibodies of different tonic signaling intensities. In some embodiments, the hybrid anti-CD20 scFv comprises (i) a light chain variable region (VL) comprising in order from amino-proximal to carboxy-proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); and (ii) a heavy chain variable region (VH) comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity -determini ng region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity-determining region 3(HCDR3), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDRl, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, or SEQ ID NOs: 15, 16, 17, 18, 20, 9 and 21 respectively, and wherein the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs:11, 12, 13, 14, 5, 6, and 19, respectively. In some embodiments, the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, and the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7 respectively. In some embodiments, the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 15, 16, 17, 18, 20, 9 and 21, respectively, and the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs: 11, 12, 13, 14, 5, 6, and 19 respectively.

[0008] In one embodiment, the genetically engineered immune cells employed in the above uses or methods are genetically engineered T cells. In some embodiments, the T cells are autologous to the patient. In other embodiments, the T cells are allogeneic to the patient. In some embodiments, the composition comprising the genetically engineered immune cells is administered to the patient intravenously. In other embodiments, the composition comprising the genetically engineered immune cells is administered to the patient by intrathecal or intraventricular administration.

[0009] In one embodiment, the VL of the hybrid anti-CD20 scFv employed in the above uses or methods comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:3, and the VH comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NON. In some embodiments, the VL comprises an amino acid sequence having the sequence of SEQ ID NO:1 or SEQ ID NO:3, and the VH comprises an amino acid sequence having the sequence of SEQ ID NO:2 or SEQ ID NON. In some embodiments, the VL comprises an amino acid sequence having the sequence of SEQ ID NO:1, and the VH comprises an amino acid sequence having the sequence of SEQ ID NON. In other embodiments, the VL comprises an amino acid sequence having the sequence of SEQ ID NON, and the VH comprises an amino acid sequence having the sequence of SEQ ID NON.

[0010] In some embodiments, the VH is amino proximal to the VL. In other embodiments, the VH is carboxy proximal to the VL.

[0011] In some embodiments, the CAR employed in the above uses or methods comprises a scFv disclosed herein, a transmembrane domain and a cytoplasmic region comprising an intracellular signaling domain. In some embodiments, the CAR further comprises a torsional linker between the transmembrane domain and the cytoplasmic region. In some embodiments, the torsional linker comprises 1, 2, 3, or 4 alanine residues. In some embodiments, the CAR comprises, in order from amino proximal to carboxy proximal end, the scFv disclosed herein, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising an intracellular signaling domain, wherein the torsional linker comprises 1-4 alanine residues.

[0012] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain from T cell receptor alpha or beta chain, CD28, CD3ε (epsilon), CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154. In some embodiments, the intracellular signaling domain is derived from CD3-zeta In some embodiments, the cytoplasmic region further comprises one or more costimulatory domains. In one embodiment, the one or more costimulatory domain(s) comprise a costimulatory domain from one or more of4-1BB (CD137), CD28, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CDl la / CD18), or ICOS (CD278).

[0013] These and other aspects of the invention will be appreciated from the ensuing descriptions of the figures and detailed description of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0015] Figures 1A-1C show development of hybrid CD20-CAR T therapy for MS. PBMC from MS patient and healthy donor were transduced with a lentiviral vector co-expressing the hybrid CD20-CAR and the NGFR marker. Cells were expanded for 11 days post transduction. Fig. 1A.Comparison of the B cell and T cell frequencies in the PBMC samples by flow cytometry. The MS patient sample exhibits a higher frequency of B cells. Fig. IB. Similar CAR expression and cell expansion post hybrid CD20-CAR T cell production in MS and healthy donor cells. The hybrid CD20-CAR can be manufactured using cells from healthy donors and MS patients. Fig. 1C.Analysis of cell phenotype post hybrid CD20-CAR T cell production. Hybrid CD20-CAR T cells from healthy donors and MS patients exhibit similar phenotype.

[0016] Figures 2A-2B show hybrid CD20-CAR T cells manufactured from MS patient eliminate CD20-high and CD20-low expressing cell lines. MS hybrid CD20-CAR T cells were co-cultured with CD20 expressing cell lines in Incucyte cell killing assays. The Raji and Nalm6 cell lines used in the assay express the mKate2 marker whose increasing intensity is correlated with cell growth.Fig.2A. Level of CD20 expression in the different cell lines. Fig.2B. Incucyte cytotoxicity assays. MS hybrid CD20-CAR T specifically eliminate cells with low and high expression of CD20.

[0017] Figures 3A-3B show hybrid CD20-CAR T cells manufactured from MS patient eliminate autologous B cells. Fig.3A. MS hybrid CD20-CAR T or n on-transduced T cells were co-cultured with autologous (donor-matched) total PBMCs for 3 days. Analysis by flow cytometry shows the complete depletion of total B cells and IgG expressing B cells by the hybrid CD20-CAR T cells.Fig. 3B. MS hybrid CD20-CAR T or non-transduced T cells were co-cultured with autologous (donor-matched) purified B cells for 3 days. Analysis by flow cytometry shows the complete depletion of total B cells and IgG expressing B cells by the hybrid CD20-CAR T cells.

[0018] Figures 4A-4F show hybrid CD20-CAR T cells manufactured from MS patient eliminate autologous B cells in an in vivo model and are detected in the brain. Fig. 4A. Schematic of MS PBMC humanized model treated with CD20 CAR-T cells. Fig. 4B. Frequency of total CD45+ human cells and human B cells in the animal blood samples 6 days after injection of total MS patient PBMCs. Fig. 4C. Flow cytometry analysis of cell phenotype of control T cells and CD20 CAR T cells that were used as treatment at day 0. Fig. 4D. Frequency of human B cells (CD19+ CD20+) of total human CD45 cells in the blood of animals at different timepoints. Analysis by flow cytometry shows complete depletion of total B cells (CD 19+ CD20+) by the hybrid CD20-CAR T cells. Fig. 4E. Frequency of CD20 CAR T cells of total human CD45 cells in the blood, spleen, bone marrow and brain assessed by flow cytometry after euthanasia. Hybrid CD20 CAR T cells were detected in all organs including the brain. Fig. 4F. Frequency of human B cells(CD19+ CD20+) of total human CD45 cells in the blood, spleen and bone marrow assessed by flow cytometry after euthanasia. Analysis by flow cytometry shows the depletion of total B cells (CD 19+ CD20+) by the hybrid CD20-CAR T cells in all organs. Of note, no human B cells were detected in the brains of animals whether they were treated with control T cells or CD20 CAR T cells (data not shown). Statistical significances of control T cell treatment compared to treatment with CD20 CAR T cells are shown (unpaired t test). *p<0.05, **p<0.01, ***p<0.001.DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.

[0020] In one embodiment, the present disclosure provides a CD20-targeting chimeric antigen receptor T cell (CAR-T) therapy for autoimmune diseases. Examples of autoimmune diseases include, but are not limited to, multiple sclerosis (MS). Engineering chimeric antigen receptors (CARs) into T cells (CAR-T cell therapy) has revolutionized the field of oncology. CD 19 and / or CD20 CAR T therapy has shown astonishing and lasting effects in B-cell leukemia and lymphoma. Remarkably, these CAR-T cell therapies have been well tolerated with minimal CNS adverse events and have also been reported to treat autoimmune diseases. CD20 appears to be a preferred target for progressive MS compared to CD 19 for several reasons. First, clinical evidence suggests that CD20 may be more resistant to antigen escape than CD19. Second, anti-CD20 therapy would avoid on target CD19-mediated neurotoxicity. And lastly, CD20 CAR-T therapy would target a specific proinflammatory CD20 expressing T cell population that has been associated with the physiopathology of MS.

[0021] Compared to sub-optimal current B cell depleting therapies such as anti-CD20 monoclonal antibody therapies, the CD20-targeting CAR T cells disclosed herein would permit a more complete B cell depletion in MS patients, and thanks to their ability to cross the blood-brain barrier,the CAR T cells disclosed herein will allow eradication of disease-inducing B cells residing in the CNS.

[0022] In one embodiment, the present disclosure provides an autologous CD20-CAR T cell therapy for the treatment of progressive MS by targeting B cells both in the periphery and the CNS. In another embodiment, the present disclosure provides an allogeneic CD20-CAR T cell therapy for the treatment of progressive MS by targeting B cells both in the periphery and the CNS.

[0023] Aspects of the present disclosure relate to a polypeptide comprising a hybrid anti-CD20 single chain variable fragment (scFv), wherein the hybrid scFv comprises framework regions (FRs) and complementarity-determining regions (CDRs) that are derived from different anti-CD20 antigen binding regions or anti-CD20 antibodies. In some embodiments, the hybrid scFv comprises FRs and CDRs that are derived from anti-CD20 antigen binding regions or anti-CD20 antibodies of different tonic signaling intensities.

[0024] In one embodiment, the hybrid scFv comprises a light chain variable region (VL) comprising in order from amino-proximal to carboxy-proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determining region 3 (LCDR3), and light chain framework region 4 (LFR4); and a heavy chain variable region (VH) comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity-determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity-determining region 3 (HCDR3), and heavy chain framework region 4 (HFR4); wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS:25, 26, 27, 18, 8, 9, and 10 respectively; and wherein HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS:22, 23, 24, 14, 5, 6, and 7 respectively.

[0025] In another embodiment, the hybrid scFv comprises a light chain variable region comprisingin order from amino-proximal to carboxy-proximal end of the light chain variable region: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4; and a heavy chain variable region comprising in order from amino-proximal to carboxy-proximal end of the heavy chain variable region: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4; wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS: 15, 16, 17, 18, 20, 9 and 21 respectively; and wherein HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOS:11, 12, 13, 14, 5, 6, and 19 respectively.

[0026] In one embodiment, the hybrid scFv comprises VL comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1, and the VH comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2. In another embodiment, the hybrid scFv comprises VL comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3, and the VH comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4.

[0027] In one embodiment, the hybrid scFv comprises VL comprising an amino acid sequence having the sequence of SEQ ID NO:1, and the VH comprises an amino acid sequence having the sequence of SEQ ID NO:2. In another embodiment, the hybrid scFv comprises VL comprising an amino acid sequence having the sequence of SEQ ID NO:3, and the VH comprises an amino acid sequence having the sequence of SEQ ID NO:4.

[0028] Aspects of the disclosure also relate to a polypeptide comprising a CAR comprising, in order from amino proximal to carboxy proximal end, a hybrid scFv comprising a variable heavy (VH) and a variable light (VL) region as disclosed herein, a transmembrane domain, and a cytoplasmic region comprising an intracellular signaling domain. In another embodiment, the CAR disclosed herein comprises, in order from amino proximal to carboxy proximal end, a hybrid scFv disclosed herein, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising an intracellular signaling domain, wherein the torsional linker comprises 1-4 alanine residues.

[0029] As used herein, “single-chain Fv” or “scFv” antibody fragments comprise at least a portion of the VH and VL domains of an antibody, such as the CDRs of each, wherein these domains are present in a single polypeptide chain. It is contemplated that an scFv includes a CDR1, CDR2, and CDR3 of a heavy chain variable region and a CDR1, CDR2, and CDR3 of a light chain variable region in some embodiments. It is further contemplated that a CDR1, CDR2, or CDR3 may comprise or consist of a sequence set forth in a SEQ ID NO provided herein as CDR1, CDR2, or CDR3, respectively. A CDR may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23, or more contiguous amino acid residues (or any range derivable therein) flanking one or both sides of a particular CDR sequence; therefore, there may be one or more additional amino acids at the N-terminal or C-terminal end of a particular CDR sequence disclosed herein. It is further contemplated that a scFv includes a HFR1, HFR2, HFR3, and / or HFR4 of a heavy chain variable region and a LFR1, LFR2, LFR3, and / or LFR4 of a light chain variable region in some embodiments. The FRs are the regions flanking the CDRs. A FR may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous amino acid residues (or any range derivable therein) flanking one or both sides of a particular FR sequence; therefore, there may be one or more additional amino acids at the N-terminal or C-terminal end of a particular FR sequence disclosed herein.

[0030] As used herein, the term “hybrid scFv” refers to a scFv that has the LFR1, LFR2, LFR3, LFR4, HFR1, HFR2, HFR3, and HFR4 from one antigen binding region or antibody, such as an antigen binding region of a scFv, antibody, nanobody, or other antibody-derived antigen-binding fragment, and has the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 from a different antigen binding region or antibody, such as an antigen binding region of a scFv, antibody, nanobody, or other antibody-derived antigen binding fragment. In one embodiment, the LFR1, LFR2, LFR3, LFR4, HFR1, HFR2, HFR3, and HFR4 are from a first anti-CD20 antigen binding region, and the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are from a second anti-CD20 antigen binding region. Accordingly, the CDR regions of one antigen binding molecule are grafted, in their corresponding order, onto the FR of another antigen binding molecule. This is exemplified by the embodiments herein that demonstrate the combining of the FR of the VH and VL regions of one antibody with the CDRs of the VH and VL regions of another antibody. The CDRs and FRs need not be derived directly from an antibody, but can be derived from any antigenbinding fragment comprising a VH and VL region, such as another scFv, a nanobody, a TCR, orother antigen binding regions known in the art and described herein. The hybrid scFv may further comprise, comprise at most, or comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions (or any range derivable therein) in the FR or CDRs of the hybrid scFv.

[0031] The hybrid scFvs of the disclosure are hybrids of two different antigen binding regions or antibodies that bind to the same antigen. In some embodiments, the two different antigen binding regions or antibodies bind to different epitopes on the same antigen. In some embodiments, the two different antigen binding regions or antibodies bind to overlapping epitopes on the same antigen. In some embodiments, two different antigen binding regions or antibodies bind to the same epitope on the antigen. The hybrid scFv may be a hybrid of two different antigen binding regions or antibodies from the same species. In some embodiments, the CDRs are derived from an antigen binding region or antibody that is from a human antibody and the FRs are derived from an antigen binding region or antibody that is also from a human antibody. In some embodiments, the CDRs are derived from an antigen binding region or antibody that is from a non-human antibody and the FRs are derived from an antigen binding region or antibody that is also from a non-human antibody. In some embodiments, the CDRs are derived from an antigen binding region or antibody that is from a mouse antibody and the FRs are derived from an antigen binding region or antibody that is also from a mouse antibody. In one embodiment, the CDRs are derived from an antigen binding region or antibody that is from a human or humanized antibody or antigen binding region and the FRs are derived from an antigen binding region or antibody that is of non-human origin, such as a mouse. In some embodiments, the hybrid scFv is derived from antigen binding regions or antibodies that have been determined to have reduced, low, or non-significant immunogenicity. In some embodiments, the hybrid scFv is derived from antigen binding regions or antibodies that have been approved for human use. In some embodiments, hybrid scFvs of the disclosure exclude humanized scFvs. Other suitable sources of the antigen binding regions or antibodies include goat, rat, horse, rabbit, mammalian, and non-human primates. In some embodiments, the CDRs and / or FRs are non-immunogenic or are reduced in immunogenicity in humans. In some embodiments, the immunogenic potential in humans of the hybrid scFv and / or polypeptide comprising the hybrid scFv is not statistically different than the non-hybrid scFv and / or polypeptide comprising the nonhybrid scFv. The non-hybrid scFv may be the scFv or antigen binding region that serves as the source of CDRs in the hybrid scFv or the non-hybrid scFv may be the scFv or antigen binding region that serves as the source of FRs in the hybrid scFv. The CDRs of the hyrid scFv maycomprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) substitutions, deletions, or additions relative to the CDRs of the antibody or antigen binding region from which the CDRs are derived from. The FRs of the hyrid scFv may comprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) substitutions, deletions, or additions relative to the FRs of the antibody or antigen binding region from which the FRs are derived from.

[0032] In some embodiments, there are polypeptides comprised of an FR from one antibody or antigen binding region and CDRs from a different antibody or antigen binding region, where the difference is because of sequence and not because they recognize / bind to different epitopes and / or antigen. The FRs and CDRs may be derived from antigen binding regions or antibodies of different tonic signaling intensities. In some embodiments, the FRs are derived from an antigen binding region or antibody of higher tonic signaling intensity than the antigen binding region or antibody in which the CDRs are derived from. In some embodiments, the CDRs are derived from an antigen binding region or antibody of higher tonic signaling intensity than the antigen binding region or antibody in which the FRs are derived from. The term “tonic signaling intensity” or “tonic signaling” when used herein refers to the level of stimulation of CAR signaling in the absence of antigen stimulation. Tonic signaling can be determined by methods generally known in the art, for example, a CTV dilution assay and / or antigen-independent activation-marker expression evaluation by antibody staining for activation markers, such as CD137 and / or CTLA-4.

[0033] In some embodiments, the hybrid scFv comprises a linker between the VH and VL. Various linkers suitable for use in scFv are generally known in the art, for example see description in US 2023 / 0084763, which is incorporated herein by reference in its entirety.

[0034] In some embodiments, the VH is amino proximal to the VL. In some embodiments, the VH is carboxy proximal to the VL. A first region is carboxy proximal to a second region when the first region is attached to the carboxy terminus of the second region. There may be further intervening amino acid residues between the first and second regions. Thus, the regions need not be immediately adjacent, unless specifically specified as not having intervening amino acid residues. The term “amino-proximal” is similarly defined in that a first region is amino-proximal to a second region when the first region is attached to the amino terminus of the second region.Similarly, there may be further intervening amino acid residues between the first and second regions unless stated otherwise.

[0035] In some embodiments, the present disclosure provides a chimeric antigen receptor (CAR) comprising the hybrid scFv described herein, and other domains generally known in the art such as a transmembrane domain and a cytoplasmic region comprising an intracellular signaling domain and costimulatory domain. For example, see description in US 2023 / 0084763. Some CAR molecules have a spacer that is between the extracellular domain and the transmembrane domain. Furthermore, one or more linkers may be included in CAR molecules between or within one or more regions, such as between different binding regions within the extracellular domain or within a binding region, such as between the variable region of a light chain (VH) and the variable region of a heavy chain (VL). In some embodiments, the polypeptide comprises a single transmembrane domain and / or a single cytoplasmic region comprising a primary intracellular signaling domain. In some embodiments, the CAR comprises, in order from amino proximal end to carboxy proximal end, the hybrid scFv, the transmembrane domain, and the cytoplasmic region comprising a primary intracellular signaling domain. In some embodiments, the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv. Examples of extracellular spacer generally known in the art include, but are not limited to, an IgG4 hinge, a CD8a hinge, an IgGl hinge, a CD34 hinge, or fragments thereof. For example, see description in US 2023 / 0084763.

[0036] Examples of transmembrane domain include, but are not limited to, an alpha or beta chain of the T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154 transmembrane domain. In some embodiments, the transmembrane domain comprises or is a CD28 transmembrane domain or is derived from a CD28 transmembrane domain. For example, see description in US 2023 / 0084763.

[0037] In some embodiments, the intracellular signaling domain is or comprises CD3-zeta or is derived from the intracellular signaling domain of CD3-zeta. Examples of CD3-zeta intracellular signaling domain are known in the art, e.g., see description in US 2023 / 0084763.

[0038] In some embodiments, the cytoplasmic region further comprises one or more costimulatory domains. In some embodiments, the cytoplasmic region comprises two costimulatory domains. Examples of costimulatory domain include, but are not limited to, costimulatory domain from 4-IBB (CD 137), CD28, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD1 la / CD18),and / or ICOS (CD278). In some embodiments, the one or more costimulatory domains comprise a costimulatory domain from CD28 or a costimulatory domain derived from CD28. For example, see description in US 2023 / 0084763.

[0039] In some embodiments, the CAR further comprises a torsional linker between the transmembrane domain and the cytoplasmic region. In some embodiments, the torsional linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues (or any derivable range therein). In some embodiments, the amino acid residues comprise or consist of alanine residues. In some embodiments, the torsional linker comprises at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein) alanine residues. In some embodiments, the torsional linker comprises at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein) contiguous alanine residues. In some embodiments, the torsional linker comprises 2 alanine residues. In some embodiments, the torsional linker comprises 3 alanine residues. In some embodiments, the torsional linker comprises 4 alanine residues. In some embodiments, the torsional linker consists of 2 alanine residues.

[0040] In other embodiments, there is a cell or a population of cells comprising a nucleic acid that encodes all or part of any polypeptide discussed herein. In certain embodiments, a cell or population of cells contains within its genome a sequence encoding any of the polypeptides described herein. This includes, but is not limited to, a lentivirus or retrovirus that has integrated into the cell’s genome. In some embodiments, a cell or population of cells expresses all or part of any CAR discussed herein. Progeny (FI, F2, and beyond) of cells in which a nucleic acid encoding a polypeptide was introduced are included in the cells or populations of cells disclosed herein. In some embodiments, a cell or population of cells comprises T cell, natural killer (NK) cell, natural killer T cell (NKT), invariant natural killer T cell (iNKT), stem cell, lymphoid progenitor cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem and progenitor cell (HSPC), hematopoietic stem cell (HSC), CD34+ cell, peripheral blood stem cell (PBSC), bone marrow cell, fetal liver cell, embryonic stem cell, cord blood cell, or induced pluripotent stem cell (iPS cell). In some embodiments, the T cell comprises a naive memory T cell. In some embodiments, the naive memory T cell comprises a CD4+ or CD8+ T cell. In some embodiments, the cells are a population of cells comprising both CD4+ and CD8+ T cells. In some embodiments, the cells are a population of cells comprising naive memory T cells comprising CD4+ and CD8+ T cells. In some embodiments, the T cell comprises a T cell from a population of CD 14 depleted, CD25 depleted,and / or CD62L enriched PBMCs.

[0041] In certain embodiments, the cells (e.g. T cells) expressing the CAR disclosed herein are autologous with respect to a patient who will receive them. In other embodiments, the cells (e g. T cells) expressing the CAR disclosed herein are not autologous and may be allogenic.

[0042] In some embodiments, the cells are engineered T cells generated from a novel three dimensional (3D) cell culture system as described in U. S. Pat. 11,154, 573, incorporated herein by reference. U. S. Pat. 11,154, 573 described methods for generating engineered T cells and compositions of the resultant T cells. In some embodiments, the T cells are non-alloreactive and express an exogenous TCR and / or CAR. These T cells are useful for “off the shelf’ T-cell therapies and do not require the use of the patient's own T cells. The novel three dimensional cell culture system can be used to produce T cells from less differentiated cells such as embryonic stem cells, pluripotent stem cells, hematopoietic stem or progenitor cells, or stem or progenitor cells generally known in the art. In certain aspects, the novel three dimensional cell culture system comprises culturing a three-dimensional cell aggregate including stroma cells and stem or progenitor cells to produce T cells, or more specifically, antigen-specific T cells or T cells that have undergone positive or negative selection in vitro. In certain embodiments, the 3D cell aggregate is cultured in a serum-free medium for a time period sufficient for the in vitro differentiation of stem or progenitor cells to T cells. In some embodiments, the T cells undergo positive selection, which provides for T cells with high avidity to specific antigens.

[0043] In some instances, the cell is a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., peripheral blood stem cell) or progenitor cell obtained from an individual.

[0044] In certain embodiments, polypeptides described throughout this disclosure are isolated, meaning they are not found in the cellular milieu. In some cases, they are purified, which means it is mostly if not completely separated from polypeptides having a different amino acid sequence and / or chemical formula.

[0045] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “oneor more than one.”

[0046] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0047] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DEFINITIONS

[0048] The peptides of the disclosure relate to peptides comprising chimeric antigen receptors, or CARs. CARs are engineered receptors, which are capable of grafting an arbitrary specificity onto an immune effector cell. In some cases, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are composed of parts from different sources.

[0049] Chimeric antigen receptors (CARs) comprise a single chain variable fragment (scFv) having a heavy chain (VH) and a light chain (VL) that each contains framework regions (FRs) flanking the complementarity determining regions (CDRs). The framework region is believed to be a major determinant of the structure of the scFv. It has been proposed that specific FR sequences, when incorporated as part of the scFv portion of the CAR, can cause tonic signaling, likely due to their effect on inducing CAR clustering. Previous work has shown that when the FR sequences of a tonically signaling CAR were combined with the CDR sequences of a non-tonically signaling CAR, the resulting hybrid CAR was shown to tonically signal. From this result, it was hypothesized that it would generally be undesirable to incorporate the FR sequences of a tonically signaling CAR in the development of new CAR molecules (Long et al. Nature Medicine, 2015.21(6):581-590). Surprisingly, the present disclosure shows that incorporation of FR from a tonically signaling CAR resulted in a CAR with superior properties. Accordingly, thecompositions and methods of the disclosure provide a redesigned anti-CD20 CAR with increased in vivo efficacy.

[0050] “Homology,” or “identity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules share sequence identity at that position. A degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 60% identity, less than 50% identity, less than 40% identity, less than 30% identity, or less than 25% identity, with one of the sequences of the current disclosure.

[0051] The terms “amino portion,” “N-terminus,” “amino terminus,” and the like as used herein are used to refer to order of the regions of the polypeptide. Furthermore, when something is N-terminal to a region it is not necessarily at the terminus (or end) of the entire polypeptide, but just at the N-terminus of the region or domain. Similarly, the terms “carboxy portion,” “C-terminus,” “carboxy terminus,” and the like as used herein is used to refer to order of the regions of the polypeptide, and when something is C-terminal to a region it is not necessarily at the terminus (or end) of the entire polypeptide, but just at the C-terminus of the region or domain.

[0052] The term “antibody” includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multi specific antibodies and antibody fragments that may be human, mouse, humanized, chimeric, or derived from another species. A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies that is being directed against a specific antigenic site.

[0053] “Antibody or functional fragment thereof means an immunoglobulin molecule that specifically binds to, or is immunologically reactive with a particular antigen or epitope, and includes both polyclonal and monoclonal antibodies. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e g., bispecific antibodies, diabodies, triabodies, and tetrabodies). The term functional antibody fragment includes antigen binding fragments or regions of antibodies, including e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments. The term scFv refers to a single chain Fv antibodyin which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.

[0054] As used herein, the term “binding affinity” refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). Binding affinity can be at least 1-fold greater, at least 2-fold greater, at least 3 -fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more (or any derivable range therein), than the binding affinity of an antibody for unrelated amino acid sequences. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0055] The term "binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

[0056] ‘ ‘Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0057] The terms “lower,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “lower,” ’’reduced,” “reduction, “decrease,” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0058] The terms “increased,” ’’increase,” “enhance,” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase,” “enhance,” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0059] Polypeptides of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., anti-CD20 antibodies). In some embodiments, an antigen binding domain comprises a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. In some embodiments, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0060] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0061] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0062] The binding affinity of the antigen binding region, such as the variable regions (heavychain and / or light chain variable region), or of the CDRs may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M. In some embodiments, the KD of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, or 10-13M (or any derivable range therein).

[0063] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SPR)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0064] In some embodiments, the polypeptide comprising the humanized binding region has equal, better, or at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104, 106, 106, 108, 109, 110, 115, or 120% binding affinity and / or expression level in host cells, compared to a polypeptide comprising a non-humanized binding region, such as a binding region from a mouse.SIGNAL PEPTIDE

[0065] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some embodiments, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g. in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0066] In some embodiments, the signal peptide is cleaved after passage of the endoplasmic reticulum, i.e., is a cleavable signal peptide. In some embodiments, a restriction site is at the carboxy end of the signal peptide to facilitate cleavage.EXTRACELLULAR SPACER

[0067] An extracellular spacer may link an antigen-binding domain to a transmembrane domain. In some embodiments, a hinge is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In one embodiment, the spacer comprises thehinge region from IgG. In some embodiments, the spacer comprises or further comprises the CH2CH3 region of immunoglobulin and portions of CD3. In some embodiments, the CH2CH3 region may have L235E / N297Q or L235D / N297Q modifications, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. In some embodiments, the spacer is from IgG4. An extracellular spacer may comprise a hinge region.

[0068] As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming interheavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U. S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0069] The length of an extracellular spacer may have effects on the CAR’s signaling activity and / or the CAR-T cells’ expansion properties in response to antigen-stimulated CAR signaling. In some embodiments, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some embodiments, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro.

[0070] As non-limiting examples, an immunoglobulin hinge region can include one of the amino acid sequences shown in WO 2023 / 0084763, which is incorporated herein by reference in its entirety.

[0071] The extracellular spacer can comprise an amino acid sequence of a human IgGl, IgG2,IgG3, or IgG4, hinge region. The extracellular spacer may also include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr.

[0072] The extracellular spacer can comprise an amino acid sequence derived from human CD8. Examples of such CD8 hinge region are known in the art, e.g. see the examples disclosed in WO 2023 / 0084763.

[0073] The extracellular spacer may comprise or further comprise a CH2 region. An exemplary CH2 region is disclosed in WO 2023 / 0084763. The extracellular spacer may comprise or further comprise a CH3 region. An exemplary CH3 region is disclosed in WO 2023 / 0084763.

[0074] When the extracellular spacer comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some embodiments, the extracellular spacer consists essentially of a hinge, CH2, and / or CH3 region, meaning that the hinge, CH2, and / or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.TRANSMEMBRANE DOMAIN

[0075] Polypeptides of the present disclosure may comprise a transmembrane domain. In some embodiments, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0076] In some embodiments, the transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is interposed between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0077] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta, CD 134, or CD7.

[0078] Exemplary transmembrane domains useful in any of the embodiments of the disclosure include those disclosed in WO 2023 / 0084763.CYTOPLASMIC REGION

[0079] After antigen recognition, receptors of the present disclosure may cluster and a signal transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domains described herein are part of the cytoplasmic region. In some embodiments, the cytoplasmic region comprises an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains.

[0080] Cytoplasmic regions and / or costimulatory regions suitable for use in the polypeptides of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen binding domain. In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif known in the art. In some embodiments, the cytoplasmic region includes DAP10 / CD28 type signaling chains.

[0081] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0082] A suitable cytoplasmic region may be a ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon);CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0083] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that may be incorporated in a CAR of the disclosure:

[0084] In some embodiments, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length DAP 12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon R1 -gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length FCERIG amino acid sequence.

[0085] In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3δ; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 delta amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3s; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3-epsilon, T3e, etc ). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length CD3 epsilon amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3y, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 gamma amino acid sequence. In some embodiments, the cytoplasmic region is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3^, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 zeta amino acid sequence.

[0086] In some embodiments, the cytoplasmic region is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some embodiments, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD79A amino acid sequence.

[0087] Specific exemplary cytoplasmic regions are known in the art, e.g. see WO 2023 / 0084763. CO STIMULATORY REGION

[0088] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0089] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVTD1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0090] Specific exemplary co-stimulatory domains are known in the art, e.g. see WO 2023 / 0084763.PEPTIDE LINKERS

[0091] In some embodiments, the polypeptides of the disclosure include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain / regions described herein. As an example, a linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, flanking the costimulatory region or on the N- or C- region of the costimulatory region, and / or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0092] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. Examples of linkers or flexible linkers are generally known in the art, e.g. see WO 2023 / 0084763.

[0093] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.THERAPEUTIC USES OR METHODS

[0094] The present disclosure includes uses or methods for treating disease and modulating immune responses in a subject in need thereof. The disclosure includes cells that may be in theform of a pharmaceutical composition that can be used to induce or modify an immune response.

[0095] Administration of the compositions according to the current disclosure will typically be via any common route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, orally, transdermally, intramuscular, intraperitoneal, intraperitoneally, intraorbitally, by implantation, by inhalation, intraventricularly, intranasally or intravenous injection.

[0096] Typically, compositions and therapies of the disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0097] The manner of application may be varied widely. Any of the conventional methods for administration of pharmaceutical compositions comprising cellular components are applicable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0098] In one embodiment, the present disclosure provides use of a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) to treat an autoimmune disease. In one embodiment, the present disclosure provides a method of treating an autoimmune disease in a patient, comprising administering to the patient a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR). In some embodiments, the CAR comprises a hybrid anti-CD20 single chain variable fragment (scFv) comprising framework regions (FRs) and complementarity-determining regions (CDRs) that are derived from different antigen binding regions or antibodies that bind CD20 In other embodiments, the use or method of treating an autoimmune disease comprises administering to the patient a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR), wherein the CAR comprises a hybrid anti-CD20 single chain variable fragment (scFv) comprising framework regions (FRs) and complementarity-determining regions (CDRs) that are derived from anti-CD20 antigen binding regions or anti-CD20 antibodies with different tonic signaling intensities. In some embodiments, the CAR further comprises a torsionallinker between the transmembrane domain and the cytoplasmic region. In one embodiment, the torsional linker comprises 1, 2, 3, or 4 alanine residues. In one embodiment, the CAR comprises, in order from amino proximal to carboxy proximal end, the scFv disclosed herein, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising an intracellular signaling domain, wherein the torsional linker comprises 1-4 alanine residues.

[0099] In one embodiment, the autoimmune disease is multiple sclerosis. In some embodiments, the autoimmune diseases can be chronic lymphocytic leukemia (CLL), granulomatosis with polyangiitis (GPA) (Wegener’s granulomatosis) and microscopic polyangiitis (MPA), non-hodgkin’s lymphoma (NHL), pemphigus vulgaris, rheumatoid arthritis, Rassmussen’s encephalitis, immune thrombocytopenic purpura, or follicular lymphoma.

[0100] Other examples of autoimmune diseases or conditions include, but are not limited to, achalasia, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, arthritis, autoimmune angioedema, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, Behcet's disease, celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy, Cogan's syndrome, congenital heart block, Crohn's disease, dermatitis, dermatomyositis, discoid lupus, Dressier's syndrome, endometriosis, fibromyalgia, fibrosing alveolitis, Graves' disease, Guillain-Barre syndrome, herpes gestationis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, Kawasaki disease, Lambert-Eaton syndrome, lichen planus, lupus, Lyme disease, multiple sclerosis, myasthenia gravis, myositis, neonatal lupus, neutropenia, palindromic rheumatism, peripheral neuropathy, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, reactive arthritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, thrombocytopenic purpura, type 1 diabetes, ulcerative colitis, uveitis, vasculitis, or vitiligo.

[0101] A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., engineered T cells expressing the CAR disclosed herein, is any amount that, when used alone or in combination with anothertherapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0102] An effective amount of a composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0103] In some embodiments, it is contemplated that doses in the range from 0.1 to 250 million CAR T cells / kg can affect the desirable properties of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or 250 million CAR T cells / kg or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, multiple times a week, multiple times a month, and / or on multiple days, weeks, or months.

[0104] “Treatment” or “treating” of a subject refers to any type of intervention or processperformed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, “treatment” or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.

[0105] In some embodiments, the uses or methods described herein generally involve genetically modifying a mammalian cell with an expression vector, or a DNA, an RNA (e.g., in vitro transcribed RNA), an adeno-associated virus (AAV), lentivirus or retrovirus comprising nucleotide sequences encoding the CAR of the present disclosure or directly transferring the CAR to the cell. The cell can be an immune cell (e g., a T lymphocyte or NK cell), a stem cell, a progenitor cell, etc.

[0106] In some embodiments, the genetic modification is carried out ex vivo. For example, T cells, stem cells, or NK cells are obtained from an individual, and the cells are then genetically modified to express the CAR of the present disclosure. In some cases, the genetically modified cells are activated ex vivo. In other cases, the genetically modified cells are introduced into an individual (e.g., the individual from whom the cells are obtained), and the genetically modified cells are activated in vivo.

[0107] The present disclosure includes uses or methods for treating autoimmune diseases and / or modulating immune responses in a subject in need thereof. The disclosure includes cells (e.g. engineered T cells expressing the CAR disclosed herein) that may be in the form of a pharmaceutical composition that can be used to induce or modify an immune response. Administration of the compositions according to the current disclosure will typically be via any common route. Examples of route of administration include, but are not limited to, parenteral, orthotopic, intradermal, subcutaneous, oral, transdermal, intramuscular, intraperitoneal, intraorbital, by implantation, by inhalation, intraventricular, intranasal or intravenous injection.

[0108] Typically, compositions and therapies of the present disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immune modifying. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0109] The manner of application may be varied widely. Any of the conventional methods for administration of pharmaceutical compositions comprising cellular components are applicable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0110] In some embodiments, it will be desirable to have multiple administrations of at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations may range from 2-day to 12-week intervals, more usually from one to two-week intervals.

[0111] The pharmaceutical compositions of the current disclosure are pharmaceutically acceptable compositions. The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.

[0112] In some embodiments, the compositions of the disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions and the preparations can also be emulsified. In other embodiments, the composition comprising the genetically engineered immune cells is administered to the patient by intrathecal or intraventricular administration.

[0113] The compositions and related methods of the present disclosure, particularly administration of a composition of the disclosure may also be used in combination with other traditional therapeutics known in the art.

[0114] The therapeutic compositions and treatments disclosed herein may precede, be co-current with and / or follow another treatment or agent by intervals ranging from minutes to weeks. In embodiments where agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time does not expire between the time of each delivery, such that the therapeutic agents would still be able to exert an advantageously combined effect on thecell, tissue or organism. For example, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more agents or treatments substantially simultaneously (e.g., within less than about a minute). In other aspects, one or more therapeutic agents or treatments may be administered or provided within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks or more, and any range derivable therein, prior to and / or after administering another therapeutic agent or treatment.SEQUENCES

[0115] Examples of VH, VL, FR and CDR sequences useful in the methods and compositions of the present disclosure are provided below.EXAMPLES

[0116] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. The Examples should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications, and GenBank Accession numbers as cited throughout this application) are hereby expressly incorporated by reference. When definitions of terms in documents that are incorporated by reference herein conflict with those used herein, thedefinitions used herein govern.Example 1Uses of Torsionally Modified CARs in Treatment for Autoimmune Diseases

[0117] The present disclosure describes a torsionally-modified hybrid anti-CD20 CAR, designated as RFR-LCDR-AA, indicating the anti-CD20 scFv comprises framework regions derived from the antibody RITUXIMAB, and CDRs derived from the Leu 16 antibody; furthermore, there are two alanine resides inserted between the transmembrane domain and the cytoplasmic domain of the CAR. It has been shown that the tuning of the CAR by the insertion of alanines induced a conformation change that significantly improved the control of tumor xenografts (see US 2023 / 0084763). ScFv hybridization in combination with this torsional reorientation in the CAR protein further enhance anti-tumor CAR-T cell function in vivo. It promotes the formation of memory T cells that can mount robust effector functions while maintaining relatively low metabolic activity levels as well as long-term persistence in vivo. Id.MATERIALS AND METHODS

[0118] For proof-of-concept studies of the autologous treatment of MS patients with hybrid CD20-CAR T cells, a lentiviral vector co-expressing the hybrid CD20-CAR and the NGFR marker was constructed, and hybrid CD20-CAR T cells were generated from MS patient. Commercially available MS patient peripheral blood Mononuclear cells (PBMCs) were obtained and transduced with the lentiviral vector 24h post T cell activation with CD3 / CD28 magnetic beads. T cells were expanded using a standard protocol (10-14 days) and CAR-expression (NGFR+ cells) was verified 5 days post transduction. CAR T cells were purified using a NGFR selection kit. Cell phenotype was monitored during the process using flow cytometry (Figure 1). Cell expansion was calculated from cell counts at the beginning and the end of the experiment. To evaluate the feasibility of manufacturing the hybrid CD20-CAR in MS patient cells, these experiments were performed in comparison to healthy donor cells (Figure 1).

[0119] To assess the potency of the MS patient hybrid CD20-CAR T cells, cytotoxicity assays (Incucyte) were performed in which the CAR T cells were co-cultured with cell lines expressing different levels of CD20. The following cell lines were used: Raji (high expression of CD20), Raji-CD20KO (no expression of CD20) and Nalm6 (low expression of CD20). These cell lines express the mKate2 marker and their growth was monitored by the live detection of the mKate markerintensity every 2 hours over 3 days. This intensity increases with cell growth and decreases with cell death (i.e. when cells were lysed by the CAR-T cells) (Figure 2). Non-transduced T cells were used as a negative control in comparison to the treatment with hybrid CD20-CAR T cells.

[0120] To mimic autologous treatment of MS patients with the hybrid CD20-CAR T cells, and to exam their ability to delete patient B cells, the CAR-T cells were co-cultured with total PBMCs from the same patient for 3 days. Cells were then harvested, and cell phenotype was analyzed by flow cytometry.

[0121] The deletion of B cells was revealed by the absence of CD20+ / CD19+ cells in the cultures. Non-transduced T cells were used as a negative control in comparison to the treatment with hybrid CD20-CAR T cells (Figure 3).

[0122] To further evaluate the ability of the MS patient hybrid CD20-CAR T cells to deplete autologous B cells, CAR-T cells were co-cultured with purified patient B cells for 3 days. Cells were then harvested, and cell phenotype was analyzed by flow cytometry. The deletion of B cells was revealed by the absence of CD20+ cells in the cultures. Non-transduced T cells were used as a negative control in comparison to the treatment with hybrid CD20-CAR T cells (Figure 3).

[0123] To assess the function of CD20 CAR T cells in an animal model, a humanized mouse model was used in which total PBMCs from an MS patient were injected in immunocompromised (NOD Scid Gamma (NSG)) mice. Eight NSG mice received i.v. injection of 10 million total MS patient PBMC (day -12). Human cell engraftment was verified 6 days later (Day -6) via flow cytometry analysis of blood samples. In the meantime, autologous CD20 CAR T cells (donor-matched) were produced ex-vivo via lentiviral transduction as described above. One million purified CD20 CAR T cells or control (non-CAR) T cells were then injected i.v. in the engrafted animals (Day 0) (Figure 4). B cell depletion and the presence of CAR T cells was evaluated in blood at day 5 and 12 via flow cytometry analysis. Spleen, bone marrow and brain cells were harvested at day 12 after animal euthanasia. Cells were analyzed by flow cytometry to assess human B cell and CAR-T cell frequency (Figure 4).RESULTS

[0124] While the frequency of B cells in the MS patient was higher compared to the healthy donor sample (Figure 1A), the CAR expression, cell expansion and cell phenotype in the patient cellspost transduction with the hybrid CD20-CAR were comparable to those in healthy donor control cells (Figures 1B-1C). This data demonstrates that hybrid CD20-CAR T cells can be produced from MS patient samples.

[0125] When tested in cytotoxicity assays against cell lines expressing CD20 at different levels, MS donor hybrid CD20-CAR T cells showed an efficient deletion of cells with high and low expression of CD20 (Figure 2). The capacity to delete cells with low expression of CD20 is important in the case of MS because some MS patients display pathogenic proinfl ammatory T cells that express low level of CD20. Studies have shown that the elimination of these CD20 expressing T cells is correlated with better control of the disease. Of note, the Raji cell line not expressing CD20 (Raji-CD20KO) was not deleted by the MS donor CD20-CAR T cells, demonstrating that the cell killing induced by the hybrid CD20-CAR T cells is specific to the CD20 antigen (Figure 2). Altogether this data demonstrates the function and specificity of MS patient hybrid CD20-CAR T cells at killing CD20 expressing cells.

[0126] When co-cultured with autologous (donor-matched) total PBMC cells or purified B cells, hybrid CD20-CAR T cells from MS patient efficiently eliminated the B cells, including IgG expressing B cells (antibody producing B cells) (Figure 3). This data is a proof-of-concept that the hybrid CD20-CAR T cells efficiently deplete autologous B cells, including pathologic B cells from MS patients.

[0127] When injected into humanized NSG mice that were engrafted with donor-matched MS PBMC, hybrid CD20-CAR T cells from MS patient efficiently eliminated the B cells in all organs analyzed (Figure 4). B cells were depleted in the blood as early as day 5 post CD20 CAR injection (Figure 4D). Twelve days post injection, CD20 CAR T cells were detected in blood, bone marrow, spleen and induced the profound depletion of human B cells (Figure 4E and 4F). No human B cells were detected in the brains of mice treated with either control T cells or CD20 CAR T cells. However, CD20 CAR T cells were detected in the brain (Figure 4E) which demonstrate that the CD20 CAR T cells can efficiently home to the brain and therefore the CNS. Altogether, this data reveals that hybrid CD20-CAR T cells represent a B cell depletion therapy for multiple sclerosis.

[0128] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those ofskill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

What is claimed is:1 Use of a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) to treat an autoimmune disease, said CAR comprising a hybrid anti-CD20 single chain variable fragment (scFv), said hybrid scFv comprisesa light chain variable region (VL) comprising in order from amino-proximal to carboxy- proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); anda heavy chain variable region (VH) comprising in order from amino-proximal to carboxy- proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity -determini ng region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity -determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementaritydetermining region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, or SEQ ID NOs:15, 16, 17, 18, 20, 9 and 21 respectively, andwherein the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs:11, 12, 13, 14, 5, 6, and 19, respectively,2. A method of treating an autoimmune disease in a patient, comprising administering to the patient a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) comprising a hybrid anti-CD20 single chain variable fragment (scFv), said hybrid scFv comprisesa light chain variable region (VL) comprising in order from amino-proximal to carboxy- proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain frameworkregion 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); anda heavy chain variable region (VH) comprising in order from amino-proximal to carboxy- proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDRl), heavy chain framework region 2 (HFR2), heavy chain complementarity -determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementaritydetermining region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, or SEQ ID NOs:15, 16, 17, 18, 20, 9 and 21 respectively, andwherein the HFR1, HFR2, HFR3, HFR4, HCDRl, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs:11, 12, 13, 14, 5, 6, and 19, respectively.

3. The use of claim 1 or the method of claim 2, wherein the genetically engineered immune cells are genetically engineered T cells,4. The use or the method of claim 3, wherein the T cells are autologous or allogeneic to said patient.

5. The use or the method of claim 1 or 2, wherein the composition is administered to the patient intravenously, or by intrathecal or intraventricular administration.

6. The use or the method of claim 1 or 2, wherein the VL comprises an amino acid sequence having the sequence of SEQ ID NO:1 or SEQ ID NO:3, and the VH comprises an amino acid sequence having the sequence of SEQ ID NO:2 or SEQ ID NO:4.

7. The use or the method of claim 1 or 2, wherein the VH is amino proximal to the VL, or the VH is carboxy proximal to the VL,8. The use or the method of claim 1 or 2, wherein said CAR comprises said hybrid scFv, a transmembrane domain and a cytoplasmic region comprising an intracellular signaling domain.

9. The use or the method of claim 8, wherein the CAR further comprises a torsional linker between the transmembrane domain and the cytoplasmic region.

10. The use or the method of claim 9, wherein the torsional linker comprises 1, 2, 3, or 4 alanine residues.

11. The use or the method of claim 1 or 2, wherein the CAR comprises, in order from amino proximal to carboxy proximal end, said hybrid scFv, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising an intracellular signaling domain, wherein the torsional linker comprises 1-4 alanine residues.

12. The use or the method of claim 8, wherein the transmembrane domain comprises a transmembrane domain from T cell receptor alpha or beta chain, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD 137 or CD154.

13. The use or the method of claim 8, wherein the intracellular signaling domain is derived from CD3-zeta.

14. The use or the method of claim 8, wherein the cytoplasmic region further comprises one or more costimulatory domains.

15. The use or the method of claim 14, wherein the one or more costimulatory domains comprise a costimulatory domain from one or more of 4-1BB (CD137), CD28, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD1 la / CD18), or ICOS (CD278).

16. Use of a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) to treat multiple sclerosis, said CAR comprising a hybrid anti-CD20 single chain variable fragment (scFv), said hybrid scFv comprisesa light chain variable region (VL) comprising in order from amino-proximal to carboxy- proximal end of the light chain variable region: light chain framework region I (LFR1), light chain complementarity-determining region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); anda heavy chain variable region (VH) comprising in order from amino-proximal to carboxy- proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity-determining region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity -determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementaritydetermining region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, or SEQ ID NOs:15, 16, 17, 18, 20, 9 and 21 respectively, andwherein the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs:11, 12, 13, 14, 5, 6, and 19, respectively.

17. A method of treating multiple sclerosis in a patient, comprising administering to the patient a composition comprising an effective amount of genetically engineered immune cells that express an anti-CD20 chimeric antigen receptor (CAR) comprising a hybrid anti-CD20 single chain variable fragment (scFv), said hybrid scFv comprisesa light chain variable region (VL) comprising in order from amino-proximal to carboxy- proximal end of the light chain variable region: light chain framework region 1 (LFR1), light chain complementarity-determining region 1 (LCDR1 ), light chain framework region 2 (LFR2), light chain complementarity-determining region 2 (LCDR2), lightchain framework region 3 (LFR3), light chain complementarity-determining region 3 (LCDR3), and light chain framework region 4 (LFR4); anda heavy chain variable region (VH) comprising in order from amino-proximal to carboxy- proximal end of the heavy chain variable region: heavy chain framework region 1 (HFR1), heavy chain complementarity -determini ng region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity -determining region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity- determining region 3 (HCDR3 ), and heavy chain framework region 4 (HFR4), wherein the LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs:25, 26, 27, 18, 8, 9, and 10, respectively, or SEQ ID NOs:15, 16, 17, 18, 20, 9 and 21 respectively, andwherein the HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NOs:22, 23, 24, 14, 5, 6, and 7, respectively, or SEQ ID NOs:11, 12, 13, 14, 5, 6, and 19, respectively.

18. The use or the method of claim 16 or 17, wherein the genetically engineered immune cells are genetically engineered T cells.

19. The use or the method of claim 18, wherein the T cells are autologous or allogeneic to said patient,20. The use or the method of claim 16 or 17, wherein the composition is administered to the patient intravenously, or by intrathecal or intraventricular administration.

21. The use or the method of claim 16 or 17, wherein the VL comprises an amino acid sequence having the sequence of SEQ ID NO:1 or SEQ ID NO:3, and the VH comprises an amino acid sequence having the sequence of SEQ ID NO:2 or SEQ ID NO:4.

22. The use or the method of claim 16 or 17, wherein the VH is amino proximal to the VL, or the VH is carboxy proximal to the VL.

23. The use or the method of claim 16 or 17, wherein the CAR comprises, in order from amino proximal to carboxy proximal end, said hybrid scFv, a transmembrane domain, a torsional linker, and a cytoplasmic region comprising an intracellular signaling domain, wherein the torsional linker comprises 1, 2, 3, or 4 alanine residues.

24. The use or the method of claim 23, wherein the transmembrane domain comprises a transmembrane domain from T cell receptor alpha or beta chain, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154.

25. The use or the method of claim 23, wherein the intracellular signaling domain is derived from CD3-zeta26. The use or the method of claim 23, wherein the cytoplasmic region further comprises one or more costimulatory domains.

27. The use or the method of claim 26, wherein the one or more costimulatory domains comprise a costimulatory domain from one or more of 4-1BB (CD137), CD28, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD1 la / CD18), or ICOS (CD278).

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