Chimeric antigen receptors for cancer treatment and related methods and compositions

Abstract

Aspects of the present disclosure relate to novel scFv molecules that are useful for incorporation into novel chimeric antigen receptors with enhanced anti-tumor activity. A further aspect relates to a polypeptide comprising a CAR, comprising, in order from the amino-proximal to the carboxy-proximal terminus, an scFv, a transmembrane domain, a kinked linker, and a cytoplasmic region comprising a primary intracellular signaling domain, wherein the kinked linker comprises 1 to 12 alanine residues. Nucleic acids comprising sequences encoding the polypeptides of the present disclosure, vectors, e.g., lentiviral vectors, comprising nucleic acids of the present disclosure, cells comprising and / or expressing nucleic acids and / or polypeptides of the present disclosure, and cell populations comprising the cellular embodiments of the present disclosure are also described. Methods of producing cells expressing the polypeptides and methods of treating patients with the polypeptides and cellular compositions of the present disclosure are also provided.

Description

[Technical Field]

[0001] This application claims the benefit of priority under U.S. Provisional Application No. 62 / 984,139, filed 2 March 2020, and U.S. Provisional Application No. 63 / 084,138, filed 28 September 2020, which are incorporated herein by reference in their entirety.

[0002] I. Field of Invention This invention generally relates to the fields of molecular biology and immunotherapy. [Background technology]

[0003] II. Background CD20 is a clinically validated target for B-cell malignancies. While CD20-targeting chimeric antigen receptor (CAR)-T cells have been clinically tested and shown to be safe, they are not as effective as CD19 CAR-T cell therapy for B-cell lymphoma. Several previous publications have stated that the primary reason for the unique potency of CD19 CARs is the lack of tonic signaling. Specifically, it has been argued that most CARs exhibit basal signaling even in the absence of antigen stimulation, that this "tonic signaling" behavior induces premature T cell dysfunction, and that CD19 CARs are uniquely effective because they do not exhibit tonic signaling and therefore do not induce premature T cell dysfunction. Furthermore, it has been proposed that CAR tonic signaling is a result of antigen-independent clustering of CAR molecules on the T cell surface, and that this clustering behavior is a result of the specific sequencing of the scFv domain of the CAR. There is a need for improved CARs that support robust, T-cell-mediated antitumor function, which may be possible by modulating the tonic signaling behavior of CARs. [Overview of the Initiative]

[0004] Summary of Disclosure The embodiments relate to compositions comprising chimeric antigen receptors, nucleic acids encoding chimeric antigen receptors, and cells capable of expressing chimeric antigen receptors, as well as methods for manipulating and / or producing chimeric antigen receptors, and methods for using these compositions. Accordingly, the compositions and methods of the present disclosure provide processes for redesigning CARs having increased in vivo potency, and the resulting CARs which may be used in the additional methods of the present disclosure.

[0005] The aspects of this disclosure include the light chain variable region (VL), which in order from the amino proximal end to the carboxy proximal end of the light chain variable region is the light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region, which in order from the amino proximal end to the carboxy proximal end is the heavy chain framework A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising a work region 1 (HFR1), a heavy chain complementarity determination region 1 (HCDR1), a heavy chain framework region 2 (HFR2), a heavy chain complementarity determination region 2 (HCDR2), a heavy chain framework region 3 (HFR3), a heavy chain complementarity determination region 3 (HCDR3), and a heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ The present invention relates to polypeptides comprising amino acid sequences having at least 90% sequence identity with ID NOs: 29, 30, 31, 22, 12, 13, and 14; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, comprising amino acid sequences having at least 90% sequence identity with SEQ ID NOs: 26, 27, 28, 18, 9, 10, and 11.

[0006] A further aspect is a polypeptide containing anti-CD20 scFv, comprising a light chain variable region comprising LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4 in order from the amino proximal end to the carboxy proximal end of the light chain variable region; and a heavy chain variable region comprising HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4 in order from the amino proximal end to the carboxy proximal end of the heavy chain variable region, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain SEQ ID This relates to polypeptides containing amino acid sequences having at least 90% sequence identity with NO:15, 16, 17, 18, 9, 10, and 23.

[0007] Further aspects include the light chain variable region (VL), which, in order from the amino proximal end to the carboxyl proximal end of the light chain variable region, includes the light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region, which, in order from the amino proximal end to the carboxyl proximal end, includes the heavy chain framework A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising a work region 1 (HFR1), a heavy chain complementarity determination region 1 (HCDR1), a heavy chain framework region 2 (HFR2), a heavy chain complementarity determination region 2 (HCDR2), a heavy chain framework region 3 (HFR3), a heavy chain complementarity determination region 3 (HCDR3), and a heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ This relates to a polypeptide comprising amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of ID NO:5; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, comprising amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:6.

[0008] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ Polypeptides are also described that include amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of ID NO:7; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, which include amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:8.

[0009] Further aspects include the light chain variable region (VL), which, in order from the amino proximal end to the carboxyl proximal end of the light chain variable region, includes the light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region, which, in order from the amino proximal end to the carboxyl proximal end, includes the heavy chain framework A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising a work region 1 (HFR1), a heavy chain complementarity determination region 1 (HCDR1), a heavy chain framework region 2 (HFR2), a heavy chain complementarity determination region 2 (HCDR2), a heavy chain framework region 3 (HFR3), a heavy chain complementarity determination region 3 (HCDR3), and a heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ The present invention relates to polypeptides comprising amino acid sequences having at least 90% sequence identity with ID NOs: 136, 137, 138, 139, 113, 114, and 115; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, comprising amino acid sequences having at least 90% sequence identity with SEQ ID NOs: 132, 133, 134, 135, 110, 111, and 112.

[0010] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ Polypeptides are also described that include amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of ID NO:141; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, which include amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:140.

[0011] Further aspects include the light chain variable region (VL), which, in order from the amino proximal end to the carboxyl proximal end of the light chain variable region, includes the light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region, which, in order from the amino proximal end to the carboxyl proximal end, includes the heavy chain framework A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising a work region 1 (HFR1), a heavy chain complementarity determination region 1 (HCDR1), a heavy chain framework region 2 (HFR2), a heavy chain complementarity determination region 2 (HCDR2), a heavy chain framework region 3 (HFR3), a heavy chain complementarity determination region 3 (HCDR3), and a heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ The present invention relates to polypeptides comprising amino acid sequences having at least 90% sequence identity with ID NO: 120, 121, 122, 123, 129, 130, and 131; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, comprising amino acid sequences having at least 90% sequence identity with SEQ ID NO: 116, 117, 118, 119, 126, 127, and 128.

[0012] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and the heavy chain variable region includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) including a heavy chain variable region (VH) comprising region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4), wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are each SEQ Polypeptides are also described that include amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of ID NO:144; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3, respectively, which include amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:143.

[0013] Aspects of this disclosure also relate to polypeptides comprising an scFv including a variable heavy chain (VH) region and a variable light chain (VL) region in order from the amino proximal terminus to the carboxyl proximal terminus, and a CAR including a transmembrane domain and a cytoplasmic region including a primary intracellular signaling domain, wherein the scFv is a hybrid scFv including a framework region (FR) and a complementarity-determining region (CDR) derived from different antigen-binding regions or antibodies that bind to the same antigen.

[0014] A further aspect relates to a polypeptide comprising a CAR, which includes, in order from the amino proximal terminus to the carboxyl proximal terminus, an scFv, a transmembrane domain, a twisted linker, and a cytoplasmic region containing a primary intracellular signaling domain, wherein the twisted linker contains 1 to 12 alanine residues.

[0015] Aspects of this disclosure relate to methods for producing a CAR, comprising the step of introducing a twisted linker between the transmembrane domain and the cytoplasmic domain of the CAR, wherein the twisted linker comprises 1 to 12 alanine residues. Also described are methods for producing a CAR having a hybrid scFv and a cytoplasmic domain comprising a transmembrane domain and a primary intracellular signaling domain, comprising the step of combining a CDR derived from a first antigen-binding domain or antibody with a FR derived from a second antigen-binding domain or antibody, wherein the first and second antigen-binding domains or antibodies bind to the same antigen. Methods for producing these CARs may further include the step of growing cells containing nucleic acids encoding one or more of these designed CARs. In some embodiments, the CAR comprises FRs and CDRs derived from antibodies or antigen-binding domains that differ in tonic signaling intensity.

[0016] Also described are nucleic acids comprising sequences encoding the polypeptides of the Disclosure, vectors comprising the nucleic acids of the Disclosure, such as lentiviral vectors, cells comprising and / or expressing the nucleic acids and / or polypeptides of the Disclosure, and cell populations comprising the cell embodiments of the Disclosure. Further aspects relate to compositions and pharmaceutically acceptable formulations comprising the polypeptides, nucleic acids, cells, and cell populations of the Disclosure. Methods for producing cells expressing the polypeptides are also provided, comprising the step of introducing the nucleic acids of the Disclosure into cells. Cells, polypeptides, and CARs produced by the methods of the Disclosure are also described. Further aspects relate to methods for treating patients with cancer, comprising the step of administering an effective amount of the composition of the Disclosure to the patient.

[0017] Further aspects include providing a CAR having a hybrid scFv and a cytoplasmic region comprising a transmembrane domain and a primary intracellular signaling domain, comprising the steps of combining a CDR derived from a first antigen-binding region or antibody with a FR derived from a second antigen-binding region or antibody, wherein the first and second antigen-binding regions or antibodies bind to the same antigen; and a method for screening a CAR, comprising the steps of evaluating, determining, or measuring the function of the CAR.

[0018] In some embodiments, the step of evaluating, determining, or measuring the function of a CAR includes evaluating the tonic signaling intensity of the CAR. In some embodiments, the step of evaluating, determining, or measuring the tonic signaling intensity includes expression evaluation by CDV dilution assay and / or antibody staining for an activation marker. In some embodiments, the step of evaluating, determining, or measuring the function of a CAR includes determining one or more of the following: tumor killing efficacy, tumor clearance efficacy, in vivo survival rate, in vivo tumor clearance, and in vivo tumor load reduction. In some embodiments, the method may further include the step of constructing a CAR comprising a hybrid scFv, which comprises combining a CDR derived from a first antigen-binding region or antibody with a FR derived from a second antigen-binding region or antibody, wherein the first and second antigen-binding regions or antibodies bind to the same antigen.

[0019] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are, respectively, SEQ ID The variable region of NO:141 contains amino acid sequences having sequence identity of at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that); HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 respectively, SEQ ID The sequence contains amino acid sequences having sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of NO:140 by at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0020] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 are, respectively, SEQ ID The variable region of NO:144 contains amino acid sequences having sequence identity of at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that); HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 are each SEQ ID The sequence contains amino acid sequences having sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of NO:143 by at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0021] A “single-chain Fv” or “scFv” antibody fragment comprises at least a portion of the VH and VL domains of the antibody, for example, each CDR, where these domains reside within a single polypeptide chain. In some embodiments, the scFv is intended to comprise heavy chain variable regions CDR1, CDR2, and / or CDR3, as well as light chain variable regions CDR1, CDR2, and / or CDR3. It is further intended that CDR1, CDR2, or CDR3 comprises, or may consist of, sequences described in the SEQ ID NOs provided herein as CDR1, CDR2, or CDR3, respectively. A CDR may contain 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 (or any derivable range thereof) consecutive amino acid residues adjacent to one or both sides of a particular CDR sequence; therefore, one or more additional amino acids may be present at the N-terminus or C-terminus of a particular CDR sequence, e.g., those shown in SEQ ID NO: 9-14, 23, 24, 25, 110-115, and 126-131. In some embodiments, scFv is further intended to include HFR1, HFR2, HFR3, and / or HFR4 of the heavy chain variable region, as well as LFR1, LFR2, LFR3, and / or LFR4 of the light chain variable region. FR is a region adjacent to the CDR. For example, scFv may include a heavy chain variable region containing HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4 from the amino terminus to the carboxy terminus. Similarly, scFv may include a light chain variable region containing LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4 from the amino terminus to the carboxy terminus. It is further intended that HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR2, or LFR4 each contain or consist of sequences described in the SEQ ID NOs provided herein as HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR2, or LFR4, respectively.FR may include 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 (or any derivable range) consecutive amino acid residues adjacent to one or both sides of a particular FR sequence; therefore, one or more additional amino acids may be present at the N-terminus or C-terminus of a particular FR sequence, for example, those shown in SEQ ID NO: 15-22, 26-31, 116-123, and 132-139.

[0022] In some embodiments, LFR1 is SEQ ID NO: 29 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR2 is SEQ ID NO: 30 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR3 is SEQ ID NO:31 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR4 is SEQ ID NO:22 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR1 is SEQ ID NO:12 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR2 is SEQ ID NO:13 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR3 is SEQ ID NO: 14 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR1 is SEQ ID NO:26 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR2 is SEQ ID NO:27 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those). In some embodiments, HFR3 is SEQ ID NO: 28 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR4 is SEQ ID NO: 18 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR1 is SEQ ID NO: 9 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR2 is SEQ ID NO: 10 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR3 is SEQ ID NO: 11 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0023] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each contain amino acid sequences with SEQ ID NO: 29, 30, 31, 22, 12, 13, and 14, respectively, and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each contain amino acid sequences with SEQ ID NO: 26, 27, 28, 18, 9, 10, and 11, respectively. In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each contain amino acid sequences with SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25, respectively; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each contain amino acid sequences with SEQ ID NO: 15, 16, 17, 18, 9, 10, and 23, respectively. In some embodiments, VL contains amino acid sequence with SEQ ID NO: 5, and VH contains amino acid sequence with SEQ ID NO: 6. In further embodiments, VL contains amino acid sequence with SEQ ID NO: 7, and VH contains amino acid sequence with SEQ ID NO: 8.

[0024] In some embodiments, LFR1 is SEQ ID NO:19 and at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, many Also 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, exactly 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 6 It contains amino acid sequences with sequence identity of 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR2 is SEQ ID NO: 20 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR3 is SEQ ID NO:21 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR4 is SEQ ID NO:22 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR1 is SEQ ID NO:24 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, It contains amino acid sequences with sequence identity of 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR2 is SEQ ID NO:13 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR3 is SEQ ID NO:25 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR1 is SEQ ID NO:15 and at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, and at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 It contains amino acid sequences having sequence identity of 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR2 is SEQ ID NO: 16 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR3 is SEQ ID NO:17 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, It contains amino acid sequences with sequence identity of 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those). In some embodiments, HFR4 is SEQ ID NO:18 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, It contains amino acid sequences with sequence identity of 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR1 is SEQ ID NO: 9 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR2 is SEQ ID NO: 10 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR3 is SEQ ID NO: 23 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0025] In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6. In some embodiments, the light chain variable region includes SEQ ID NO: 5 and at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 6 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 It contains amino acid sequences having sequence identity of 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those), and the heavy chain variable region is SEQ IDNO: 6 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0026] In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8. In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8. NO:7 and at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 It contains amino acid sequences having sequence identity of 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that), and the heavy chain variable region is SEQ IDNO:8 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0027] In some embodiments, LFR1 is SEQ ID NO: 136 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR2 is SEQ ID NO:137 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR3 is SEQ ID NO: 138 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR4 is SEQ ID NO: 139 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR1 is SEQ ID NO:113 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR2 is SEQ ID NO: 114 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR3 is SEQ ID NO: 115 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR1 is SEQ ID NO: 132 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR2 is SEQ ID NO: 133 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR3 is SEQ ID NO: 134 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR4 is SEQ ID NO: 135 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR1 is SEQ ID NO: 110 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 6 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 6 Amino acids having sequence identity of 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range). Includes sequences. In some embodiments, HCDR2 includes SEQ ID NO: 111 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR3 is SEQ ID NO: 112 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0028] In some embodiments, LFR1 is SEQ ID NO: 120 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR2 is SEQ ID NO: 121 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR3 is SEQ ID NO: 122 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LFR4 is SEQ ID NO: 123 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR1 is SEQ ID NO: 129 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR2 is SEQ ID NO: 130 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, LCDR3 is SEQ ID NO: 131 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR1 is SEQ ID NO: 116 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR2 is SEQ ID NO: 117 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR3 is SEQ ID NO: 118 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HFR4 is SEQ ID NO: 119 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR1 is SEQ ID NO: 126 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 6 3, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 6 Amino acids having sequence identity of 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range). Includes sequences. In some embodiments, HCDR2 includes SEQ ID NO: 127 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).In some embodiments, HCDR3 is SEQ ID NO: 128 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0029] In some aspects, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each contain amino acid sequences with SEQ ID NO: 136, 137, 138, 139, 113, 114, and 115, respectively, and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each contain amino acid sequences with SEQ ID NO: 132, 133, 134, 135, 110, 111, and 112, respectively. In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each contain amino acid sequences with SEQ ID NO: 120, 121, 122, 123, 129, 130, and 131, respectively; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each contain amino acid sequences with SEQ ID NO: 116, 117, 118, 119, 126, 127, and 128, respectively. In some embodiments, VL contains amino acid sequence with SEQ ID NO: 141, and VH contains amino acid sequence with SEQ ID NO: 140. In further embodiments, VL contains amino acid sequence with SEQ ID NO: 144, and VH contains amino acid sequence with SEQ ID NO: 143.

[0030] In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:141, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:140. In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:141, and at least 60, 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 The sequence contains amino acid sequences with sequence identity of 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range), and the heavy chain variable region is SEQ IDNO: 140 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0031] In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:144, and the heavy chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:143. In some embodiments, the light chain variable region includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:144, and at least 60, 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 The sequence contains amino acid sequences with sequence identity of 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range), and the heavy chain variable region is SEQ IDNO:143 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at least 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67 It contains amino acid sequences having sequence identity of 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within those).

[0032] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:5, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:5.

[0033] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:7, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:8.

[0034] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:141, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:140.

[0035] In some embodiments, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:144, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:143.

[0036] The scFv of this disclosure may be a hybrid scFv. The term "hybrid" refers to an scFv having LFR1, LFR2, LFR3, LFR4, HFR1, HFR2, HFR3, and HFR4 derived from a certain antigen-binding region or antibody, e.g., scFv, antibody, antigen-binding region of a nanobody, or other antibody-derived antigen-binding fragment, and having LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 derived from a different antigen-binding region or antibody, e.g., scFv, antibody, antigen-binding region of a nanobody, or other antibody-derived antigen-binding fragment. In a hybrid scFv, LFR1, LFR2, LFR3, LFR4, HFR1, HFR2, HFR3, and HFR4 are derived from a first antigen-binding region, and LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are derived from a second antigen-binding region. Therefore, the CDR region of one antigen-binding molecule is transplanted onto the FR of another antigen-binding molecule in a corresponding order. This is further illustrated by embodiments herein demonstrating the combination of the FRs of the VH and VL regions of one antibody and the CDRs of the VH and VL regions of another antibody. The CDRs and FRs do not necessarily originate directly from the antibody, but may originate from any antigen-binding fragment containing VH and VL regions, such as another scFv, nanobody, TCR, and other antigen-binding regions known in the art and described herein. The hybrid may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions (or any derivable range thereof) in the FR or CDR of the hybrid CAR. The hybrid scFv of this disclosure is a hybrid 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, 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. A hybrid scFv may be a hybrid of two different antigen-binding regions or antibodies derived from the same species. In some embodiments, the CDR is derived from an antigen-binding region or antibody derived from a human antibody, and the FR is also derived from an antigen-binding region or antibody derived from a human antibody. In some embodiments, the CDR is derived from an antigen-binding region or antibody derived from a non-human antibody, and the FR is also derived from an antigen-binding region or antibody derived from a non-human antibody. In some embodiments, the CDR is derived from an antigen-binding region or antibody derived from a mouse antibody, and the FR is also derived from an antigen-binding region or antibody derived from a mouse antibody. In one embodiment, the CDR is derived from an antigen-binding region or antibody derived from a human or humanized antibody or antigen-binding region, and the FR is derived from a non-human, e.g., mouse antigen-binding region or antibody. In some embodiments, a hybrid scFv is derived from an antigen-binding region or antibody that has been determined to have reduced, low, or insignificant immunogenicity. In some embodiments, the hybrid scFv is derived from an antigen-binding region or antibody approved for use in humans. In some embodiments, the hybrid scFv of this disclosure excludes humanized scFv. Other suitable sources of antigen-binding regions or antibodies include goats, rats, horses, rabbits, mammals, and non-human primates. In some embodiments, the CDR and / or FR are non-immunogenic or have reduced immunogenicity in humans. In some embodiments, the potential for immunogenicity in humans of the hybrid scFv and / or polypeptides containing the hybrid scFv is not statistically different from that of the non-hybrid scFv and / or polypeptides containing the non-hybrid scFv. The non-hybrid scFv may be an scFv or antigen-binding region that serves as the source of the CDR of the hybrid scFv, or the non-hybrid scFv may be an scFv or antigen-binding region that serves as the source of the FR of the hybrid scFv.The CDR of a hybrid scFv has 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 CDRs, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, compared to the CDR of the antibody or antigen-binding region from which the CDR originates. This may include 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions, deletions, or additions, or 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 thereof) substitutions, deletions, or additions. The FRs of the hybrid scFv are 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, compared to the FRs of the antibody or antigen-binding region from which the FRs originate, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 This may include 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 substitutions, deletions, or additions, or 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 thereof) substitutions, deletions, or additions.

[0037] In some embodiments, polypeptides exist that consist of a FR derived from one antibody or antigen-binding region and a CDR derived from a different antibody or antigen-binding region, where the difference is due to sequence and not to recognition of / binding to different epitopes and / or antigens. The FR and CDR may be derived from antigen-binding regions or antibodies having different tonic signaling intensities. In some embodiments, the FR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which the CDR is derived. In some embodiments, the CDR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which the FR is derived. When used herein, the terms “tonic signaling intensity” or “tonic signaling” refer to the level of stimulation of CAR signaling in the absence of antigen stimulation. Tonic signaling can be determined by the CTV dilution assay (described in Example 3) and / or by the evaluation of antigen-independent activation marker expression by antibody staining for activation markers, e.g., CD137 and / or CTLA-4 (described in Example 3). In some embodiments, the median fluorescence intensity (MFI) in the CTV dilution assay is at least 1.5, 2, 2.5, 3, 3.5, 4, or 5-fold (or any derivable range) higher in cells with non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR or CDR of the hybrid scFv compared to hybrid scFvs. In some embodiments, CD137+ expression is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold (or any derivable range) higher in cells with hybrid scFvs compared to non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR or CDR. In some embodiments, CTLA-4+ expression is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold (or any derivable range) higher in cells with hybrid scFvs compared to non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR or CDR.Furthermore, hybrid scFv may include CDRs and FRs with different tonic signaling intensities. For example, the CDR may be derived from a CAR that exhibits at least 1.5, 2, 2.5, 3, 3.5, 4, or 5 times (or any derivable range within that range) higher MFI in a CTV dilution assay compared to the CAR from which the FR is derived. In some embodiments, the CDR may be derived from a CAR that exhibits at least 1.5, 2, 2.5, 3, 3.5, 4, or 5 times (or any derivable range within that range) higher MFI in a CTV dilution assay compared to the CAR from which the CDR is derived. In some embodiments, the CDR may be derived from a CAR that exhibits at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range within that range) higher CD137+ expression compared to the CAR from which the CDR is derived. In some embodiments, FR may be derived from a CAR that exhibits at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range) higher CD137+ expression compared to the CAR from which FR is derived. In some embodiments, CDR may be derived from a CAR that exhibits at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range) higher CTLA4+ expression compared to the CAR from which CDR is derived. In some embodiments, FR may be derived from a CAR that exhibits at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range) higher CTLA4+ expression compared to the CAR from which FR is derived. In some embodiments, the CDR may be derived from a CAR that expresses an activation marker at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range) higher than the CAR from which the CDR is derived. In some embodiments, the FR may be derived from a CAR that expresses an activation marker at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times (or any derivable range) higher than the CAR from which the FR is derived.It may be further clarified that polypeptides have increased tumor-killing activity compared to CARs containing non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR of hybrid scFvs, and / or have increased tumor-killing activity compared to CARs containing non-hybrid scFvs derived from the same antigen-binding region or antibody as the CDR of hybrid scFvs. For example, CAR T cells containing hybrid scFvs may show at least significantly lower, or at least 2, 3, 4, or 5-fold lower, target cell magnification changes compared to non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR or CDR after co-culture for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, polypeptides have increased tumor cell clearance activity compared to CARs containing non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or polypeptides have increased tumor killing activity compared to CARs containing non-hybrid scFvs derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. For example, CAR T cells containing hybrid scFvs may exhibit at least significantly more, or at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000-fold, tumor cell clearance fold change (as indicated by a decrease in the mean radiance of labeled cells) than non-hybrid scFvs derived from the same antigen-binding region or antibody as the FR or CDR, at least 20, 40, 60, 80, 100, 120, 140, 160, or 180 days after T cell injection. In some embodiments, the CDR or FR of a hybrid CAR is derived from a non-hybrid scFv of a CAR that has been shown not to have in vivo efficacy as seen by significant tumor killing activity, tumor cell clearance, or increased in vivo tumor clearance or survival compared to a negative control.

[0038] In some embodiments, the polypeptide contains a linker between VH and VL. In some embodiments, the linker is 4 to 40 amino acids long. In some embodiments, the linker is 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. Acid residue length, at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residue lengths, more than Also, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residue lengths, or approximately 4, 5, 6, 7, The amino acid residue length is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues (or any derivable range within that range). In some embodiments, the linker contains at least four glycine residues and / or serine residues. In some embodiments, the linker contains at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range thereof) glycine residues and / or serine residues. In some embodiments, the linker is (GGGGS) nThis includes, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). In some embodiments, the linker includes GSTSGGGSGGGSGGGGSS (SEQ ID NO: 32), or a linker having sequence identity with SEQ ID NO: 32 of at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein). In some embodiments, the linker includes the amino acid sequence: (EAAAK) n Includes or (EAAAK) n It consists of, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range within that range). In some embodiments, the linker includes GGGGS. In some embodiments, the linker is (GGGGS)4.

[0039] In some embodiments, VH is amino-proximal to VL. In some embodiments, VH is carboxyl-proximal to VL. When the first region is attached to the carboxyl terminus of the second region, the first region is carboxyl-proximal to the second region. Further intervening amino acid residues may be present between the first and second regions. Therefore, the regions do not need to be directly adjacent unless otherwise specified. The term "amino-proximal" is similarly defined as the first region being amino-proximal to the second region when the first region is attached to the amino terminus of the second region. Similarly, further intervening amino acid residues may be present between the first and second regions unless otherwise specified.

[0040] In some embodiments, the polypeptide comprises a chimeric antigen receptor (CAR) comprising an scFv and a cytoplasmic region containing a transmembrane domain and a primary intracellular signaling domain. In some embodiments, the CAR molecules described herein have three main regions: an extracellular domain that binds to one or more target molecules, a cytoplasmic region containing a primary intracellular signaling domain, and a transmembrane region between the extracellular and cytoplasmic domains. Some CAR molecules have a spacer between the extracellular and transmembrane domains. Furthermore, the CAR molecule may include one or more linkers between or within one or more regions, for example, between different binding regions within the extracellular domain, or within binding regions, for example, between the variable region (VH) of the light chain and the variable region (VL) of the heavy chain. Any embodiment relating to a particular region may be carried out relating to any other particular region disclosed herein. Any of these regions may be directly adjacent to either the N-terminal or C-terminal side of another region, depending on its function, but 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, 3 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 8 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, or at most, at least, or 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 It is also intended that 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 (or any derivable range thereof) amino acids may be interposed between consecutive regions. In some embodiments, the polypeptide comprises a single transmembrane domain and / or a single cytoplasmic region containing a primary intracellular signaling domain. In some embodiments, the polypeptide comprises, in order from the amino proximal terminus to the carboxyl proximal terminus, an scFv, a transmembrane domain, and a cytoplasmic region containing a primary intracellular signaling domain. In some embodiments, the CAR is monospecific. In some embodiments, the CAR is bispecific. In some embodiments, the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv. In some embodiments, the extracellular spacer is 8 to 1000 amino acids long. In some embodiments, the extracellular spacer is 8 to 500 amino acids long. In some embodiments, the extracellular spacer is 100 to 300 amino acids long. In some embodiments, the extracellular spacer has fewer than 100 amino acids. According to some accounts, extracellular spacers are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、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、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 3 48, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 37 9, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410 ,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids, at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 4 01, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 45 7, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids, precisely 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 4 10, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 44 1, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472 , 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids, or approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 4 08, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 ,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474, The amino acids are 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids (or any derivable range thereof). In some embodiments, the extracellular spacer is or comprises an IgG4 hinge, a CD8α hinge, an IgG1 hinge, a CD34 hinge, or a fragment thereof. In some embodiments, the extracellular spacer comprises an IgG4 hinge or a fragment thereof. In some embodiments, the extracellular spacer comprises or further comprises a CH1 region, a CH2 region, and / or a CH3 region. In some embodiments, the spacer comprises or comprises an IgG4 hinge polypeptide, a CH2 region, and a CH3 region. In some embodiments, the CH2 region includes an L235E substitution and / or an N297Q substitution. In some embodiments, the spacer includes a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:36. In some embodiments, the spacer includes SEQ ID NO:36 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at most 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, The polypeptide or its fragments have sequence identity of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or any derivable range within that).

[0041] In some embodiments, the transmembrane domain is the transmembrane domain of the α or β 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. In some embodiments, the transmembrane domain contains, is, or is derived from the CD28 transmembrane domain. In some embodiments, the transmembrane domain contains a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:37. In some embodiments, the transmembrane domain is SEQ ID NO:37, and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at most 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 6 The polypeptide or its fragments having sequence identity of 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0042] In some embodiments, the primary intracellular signaling domain is CD3ζ, contains CD3ζ, or is derived from an intracellular signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:39. In some embodiments, the primary intracellular signaling domain comprises SEQ ID NO:39 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at most 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 6 The polypeptide or its fragments having sequence identity of 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0043] In some embodiments, the cytoplasmic region further comprises one or more costimulatory domains. In some embodiments, the cytoplasmic region comprises two costimulatory domains. In some embodiments, one or more costimulatory domains comprise one or more costimulatory domains derived from 4-1BB(CD137), CD28, IL-15Rα, OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a / CD18), and / or ICOS(CD278). In some embodiments, one or more costimulatory domains comprise a costimulatory domain derived from CD28. In some embodiments, the costimulatory domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:38. In some embodiments, the costimulatory domain comprises SEQ ID NO:38 and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at most 60, 61, 62, 63, 64, 6 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or approximately 60, 61, 62, 63, 64, 65, 66, 67, 68, 6 The polypeptide or its fragments having sequence identity of 9, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0044] In some embodiments, the polypeptide further comprises a twisted linker between the transmembrane domain and the cytoplasmic domain. In some embodiments, the twisted linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range thereof) amino acid residues. In some embodiments, the amino acid residues comprise or consist of alanine residues. In some embodiments, the twisted linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range thereof) alanine residues. In some embodiments, the twisted linker contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues, and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range thereof). In some embodiments, the twisted linker consists of 2 or 4 alanine residues. In some embodiments, the twisted linker contains 2 alanine residues. In some embodiments, the twisted linker contains 3 alanine residues. In some embodiments, the twisted linker contains 4 alanine residues. In some embodiments, the twisted linker contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive alanine residues, and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range thereof). In some embodiments, the twisted linker consists of 2 alanine residues.

[0045] In some embodiments, the scFv includes an anti-CD20 scFv. In some embodiments, the scFv includes an anti-GD2 scFv. In some embodiments, the scFv is anti-BCMA, anti-CD123, anti-CD138, anti-CD19, anti-CD20, anti-CD22, anti-CD38, anti-CD5, anti-Igκ chain, anti-LeY, Anti-NKG2D, anti-ROR1, anti-WT1, anti-C-Met, anti-CAIX, anti-CD133, anti-CD171, anti-CD70, anti-CEA, anti-EGFR, anti-EGFRvIII, anti-Ep-CA M, anti-EphA2, anti-FAP, anti-GD2, anti-GPC3, anti-Her2, anti-HPV16-E6, anti-IL13Ra2, anti-MAGEA3, anti-MAGEA4, anti-MART1, anti-Mesosuri Includes scFvs such as Mesothlin, anti-MUC1, anti-MUC6, anti-NY-ESO-1, anti-PD-L1, anti-PSCA, anti-PSMA, anti-ROR1, or anti-VEGFR2.

[0046] Nucleic acids comprising sequences and portions thereof encoding polypeptides disclosed herein are provided in embodiments. Nucleic acids may include RNA or DNA. In certain embodiments, the nucleic acid is an expression construct. In some embodiments, the expression construct is a vector. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a retroviral vector or derived from a retrovirus. In some embodiments, the retroviral vector includes a lentiviral vector or derived from a lentivirus. Note that in certain embodiments, the viral vector is an embedded nucleic acid. Furthermore, the nucleic acid may be a molecule involved in gene editing, such that a CAR-coding nucleic acid (e.g., guide RNA) is used to embed the CAR-coding sequence into a specific locus of the genome, e.g., the TRAC gene. In some embodiments, this includes a gene editing system, e.g., CRISPR / Cas9. A nucleic acid, polynucleotide, or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of "sequence identity" or "homology" with another sequence (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or any derivable range therein) of sequence identity or homology, i.e., when aligned, that percentage of bases (or amino acids) are the same in a comparison of the two sequences. This alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, e.g., those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. A nucleic acid is intended to have such sequence identity or homology with any nucleic acid SEQ ID NO provided herein.

[0047] In other embodiments, there exist cells or cell populations containing nucleic acids encoding all or part of any polypeptides described herein. In certain embodiments, a cell or cell population contains a sequence encoding any polypeptide described herein within its genome. This includes, but is not limited to, lentiviruses or retroviruses integrated into the cell's genome. In some embodiments, a cell or cell population expresses all or part of any CAR described herein, for example, having an amino acid sequence of any of SEQ ID NO: 1 to 144, but not limited to the following. The offspring (F1, F2, and subsequent generations) of cells into which nucleic acids encoding polypeptides have been introduced are included in the cells or cell populations disclosed herein. In some embodiments, cells or cell populations include T cells, natural killer (NK) cells, natural killer T cells (NKT), invariant natural killer T cells (iNKT), stem cells, lymphocyte progenitor cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells or hematopoietic progenitor cells (HSPCs), hematopoietic stem cells (HSCs), CD34+ cells, peripheral blood stem cells (PBSCs), bone marrow cells, fetal hepatocytes, embryonic stem cells, umbilical cord blood cells, and induced pluripotent stem cells (iPS cells). Specific embodiments relate to cells that are T cells or NK cells. In some embodiments, T cells include naive memory T cells. In some embodiments, naive memory T cells include CD4+ T cells or CD8+ T cells. In some embodiments, cells are a cell population that includes both CD4+ T cells and CD8+ T cells, as well as naive memory T cells. In some embodiments, the T cells include T cells derived from a population of PBMCs that are CD14 depleted, CD25 depleted, and / or CD62L enriched.

[0048] In some aspects, the present disclosure relates to cells comprising one or more polypeptides described herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are progenitor cells or stem cells. In some embodiments, the progenitor cells or stem cells differentiate into immune cells in vitro. In some embodiments, the cells are T cells. In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, the cells are natural killer cells. In some embodiments, the cells are ex vivo. The term immune cell includes cells of the immune system involved in the body's defense against both infectious diseases and foreign substances. Immune cells can include, for example, neutrophils, eosinophils, basophils, natural killer cells, lymphocytes, such as B cells and T cells, and monocytes. T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, γδT cells, regulatory T cells, suppressor T cells, and natural killer T cells. In a specific embodiment, the T cells are regulatory T cells.

[0049] In some embodiments, the cell population comprises 10 3 to 10 8 cells. In some embodiments, the population comprises about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 cells, at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 cells, or at most about 10 2 , 10 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 It is an individual cell (or any derivable range within that). In a particular embodiment, the cell is self to the patient who receives them. In other embodiments, the cell may not be self, but an allogeneic cell.

[0050] In some aspects of this disclosure, the method aspect relates to infecting cells with a virus encoding the polypeptide of this disclosure. In some aspects, the virus includes lentiviruses, or viruses or vectors derived from lentiviruses. In some aspects, the cells are T cells, natural killer (NK) cells, natural killer T cells (NKT), invariant natural killer T cells (iNKT), stem cells, lymphocyte progenitor cells, peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal hepatocytes, embryonic stem cells, umbilical cord blood cells, and induced pluripotent stem cells (iPS cells). In some aspects, the cells are T cells or NK cells. In some aspects, the T cells include naive memory T cells. In some aspects, the naive memory T cells include CD4+ T cells or CD8+ T cells. In some aspects, the cells are not yet T cells or NK cells, and the method further includes the step of culturing the cells under conditions that promote the differentiation of the cells into T cells or NK cells.

[0051] In some embodiments, the method further includes the step of culturing cells under conditions that promote cell growth, before and / or after the introduction of nucleic acids into the cells. In some embodiments, the cells are cultured in serum-free medium.

[0052] The additional method relates to the treatment of a patient having cancer, comprising the step of administering to the patient an effective amount of a composition comprising a population of cells expressing the polypeptide of the Disclosure. In some embodiments, the patient has recurrent or recurrent cancer. Further embodiments include the step of administering the additional treatment to the patient. In some embodiments, the patient has previously received treatment for cancer. In some embodiments, the patient has been determined to be resistant to the prior treatment. The prior treatment may be a cancer therapeutic agent described herein, e.g., described as the additional treatment. Further embodiments include the step of administering chemotherapy and / or radiation to the patient. In some embodiments, the additional treatment includes immunotherapy. In some embodiments, the additional treatment includes the additional treatment described herein. In some embodiments, the immunotherapy includes immune checkpoint inhibitor therapy. In some embodiments, the immunotherapy includes the immunotherapy described herein. In some embodiments, the immune checkpoint inhibitor therapy includes PD-1 inhibitors and / or CTLA-4 inhibitors. In some embodiments, the immune checkpoint inhibitor therapy includes one or more inhibitors of one or more immune checkpoint proteins described herein.

[0053] In some embodiments, cancer includes CD20+ cancer, where CD20+ cancer contains CD20+ cells or contains at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% CD20+ cancer cells in a population of tumor cells. In some embodiments, cancer includes melanoma. The CAR polypeptides of this disclosure include all or part of the amino acid sequences described herein, and at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 8 5, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, at most 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or exactly 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 It may contain, or have a region, domain, linker, spacer, or other part thereof, an amino acid sequence that is identical to, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range thereof).In a particular embodiment, CAR polypeptide is one of SEQ ID NO: 1-144 and 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, less 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at most 50, 51, 52, 53, 5 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or exactly 50, 51, 52, 53, 54, 55, 56, 57, 5 It contains or consists of amino acid sequences that are identical 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range within that range).

[0054] Cancer may be lymphoma or neuroblastoma. In embodiments in which the CAR comprises a CD20 CAR, the targeted cancer may be lymphoma. In embodiments in which the CAR comprises a GD2 CAR, the targeted cancer may be neuroblastoma. Cancers that can be treated in the methods of this disclosure may also include cancers of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, cancer can be, but is not limited to, the following histological types: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrin-producing tumor; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and Cholangiocarcinoma; cord-like adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumors; malignant carcinoid tumors; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; mixed type of papillary and follicular adenocarcinoma Adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; ear canal carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; steroid-producing tumor (lipid cell) tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus angiosarcoma; malignant melanoma; melanin-deficient melanoma; superficial spreading melanoma; malignant melanoma of giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus;Sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müller's mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; embryonic cancer; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant periangiocarcinoma; Lymphangiosarcoma; Osteosarcoma; Osteogenic sarcoma of the proximal cortex; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloepithelial odontoma; Malignant ameloblastoma; Ameloepithelial fibrosarcoma; Malignant pineal glandoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrocystic sarcoma Astrocytoma; glioblastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectodermal; cerebellar sarcoma; gangliblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granuloma; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant small lymphocytic lymphoma; malignant large cell diffuse lymphoma; malignant Follicular lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0055] In certain embodiments, the polypeptides described throughout this disclosure are isolated, i.e., not found in the cellular environment. In some cases, they are purified, i.e., largely, if not completely, separated from polypeptides having different amino acid sequences and / or chemical formulas.

[0056] In some embodiments, this disclosure provides a method for treating a subject having cancer, comprising the step of administering to the subject an effective amount of a cell population or a pharmaceutical composition comprising a chimeric polypeptide or a nucleic acid encoding a chimeric polypeptide.

[0057] The use of one or more sequences or compositions may be utilized based on any of the methods described herein. Other embodiments are described throughout this application. Any embodiment described with respect to one aspect of this disclosure is also applicable to other aspects of this disclosure, and vice versa. For example, any step of any method described herein may be applicable to any other method. Furthermore, any method described herein may have the exclusion of any step or combination of steps. The embodiments in the Examples section are understood to be applicable to all aspects of the technology described herein.

[0058] Throughout this application, the term “approximately” is used in accordance with its plain, ordinary meaning in the fields of cell biology and molecular biology to indicate that a value includes the standard deviation of the error of the instrument or method used to determine that value.

[0059] When used with the term "includes," the use of the words "a" or "an" can mean "one," but also coincide with the meanings of "one or more," "at least one," and "one or more."

[0060] As used herein, the terms “or” and “and / or” are used to describe multiple components that are combined or mutually exclusive. For example, “x, y, and / or z” could mean “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically intended that x, y, or z may be specifically excluded from certain embodiments.

[0061] "Comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), "characterized by" (and any form of including, e.g., "characterized as"), or "containing" (and any form of containing, e.g., "contains" and "contain") are comprehensive or non-restrictive and do not exclude additional unlisted elements or steps of method.

[0062] Compositions and methods of use thereof may "contain," "essentially consist of," or "consist of" any of the components or processes disclosed throughout this Spec. The phrase "consist of" excludes elements, processes, or components not specified. The phrase "essentially consist of" limits the scope of the described subject matter to those that do not substantially affect the specified material or process and its basic and novel characteristics. Embodiments described by the term "contain" are intended to also be carried out by the terms "consist of" or "essentially consist of."

[0063] It is specifically intended that any limitation described in relation to one aspect of the present invention may also apply to any other aspect of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to prepare or utilize any composition of the present invention. Aspects of the embodiments described in the examples may also be embodiments that may be implemented in different embodiments or in the context of embodiments described elsewhere in this application, for example, in the summary of the invention, the detailed description of the embodiments, the claims, and the description of the drawings.

[0064] [Invention 1001] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 29, 30, 31, 22, 12, 13, and 14; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 26, 27, 28, 18, 9, 10, and 11. The aforementioned polypeptide. [Invention 1002] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 5; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 6. The aforementioned polypeptide. [Invention 1003] A polypeptide according to Invention 1001 or 1002, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:5, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:5. [Invention 1004] A polypeptide according to any of the inventions 1001 to 1003, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each comprise the amino acid sequences of SEQ ID NO: 29, 30, 31, 22, 12, 13, and 14; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each comprise the amino acid sequences of SEQ ID NO: 26, 27, 28, 18, 9, 10, and 11. [Invention 1005] Light chain variable regions including LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4, in order from the amino proximal end to the carboxyl proximal end; and The heavy chain variable region includes HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4, in order from the amino proximal end to the carboxyl proximal end of the heavy chain variable region. A polypeptide containing anti-CD20 scFv, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 15, 16, 17, 18, 9, 10, and 23. The aforementioned polypeptide. [Invention 1006] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:7; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:8. The aforementioned polypeptide. [Invention 1007] A polypeptide according to Invention 1005 or 1006, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO:7, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO:8. [Invention 1008] A polypeptide according to any of the Invention 1005 to 1007, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 each comprise the amino acid sequences of SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 each comprise the amino acid sequences of SEQ ID NO: 15, 16, 17, 18, 9, 10, and 23. [Invention 1009] A polypeptide according to any one of the invention 1001 to 1004, wherein the light chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, and the heavy chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6. [Invention 1010] The polypeptide of the present invention 1009, wherein VL contains the amino acid sequence of SEQ ID NO: 5 and VH contains the amino acid sequence of SEQ ID NO: 6. [Invention 1011] A polypeptide according to any one of the invention 1005 to 1008, wherein the light chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7, and the heavy chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8. [Invention 1012] The polypeptide of the present invention 1011, wherein VL contains the amino acid sequence of SEQ ID NO:7 and VH contains the amino acid sequence of SEQ ID NO:8. [Invention 1013] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 136, 137, 138, 139, 113, 114, and 115; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 132, 133, 134, 135, 110, 111, and 112. The aforementioned polypeptide. [Invention 1014] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:141; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:140. The aforementioned polypeptide. [Invention 1015] A polypeptide according to any of the inventions 1013 to 1014, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO: 141, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO: 140. [Invention 1016] A polypeptide according to any one of invention 1013 to 1015, wherein the light chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:141, and the heavy chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:140. [Invention 1017] The polypeptide of the present invention 1016, wherein VL contains the amino acid sequence of SEQ ID NO:140 and VH contains the amino acid sequence of SEQ ID NO:141. [Invention 1018] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 120, 121, 122, 123, 129, 130, and 131; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 116, 117, 118, 119, 126, 127, and 128. The aforementioned polypeptide. [Invention 1019] The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). A polypeptide comprising an anti-GD2 single-chain variable fragment (scFv) containing, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:144; HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:143. The aforementioned polypeptide. [Invention 1020] A polypeptide according to any of the inventions 1013 to 1014, wherein LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 comprises the amino acid sequences of LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and / or LCDR3 derived from the variable region of SEQ ID NO: 144, and / or HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 comprises the amino acid sequences of HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and / or HCDR3 derived from the variable region of SEQ ID NO: 143. [Invention 1021] A polypeptide according to any of invention 1018 to 1020, wherein the light chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:144, and the heavy chain variable region contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO:143. [Invention 1022] The polypeptide of the present invention 1021, wherein VL contains the amino acid sequence of SEQ ID NO:144 and VH contains the amino acid sequence of SEQ ID NO:143. [Invention 1023] A polypeptide comprising a linker between VH and VL, any of invention 1001 to 1022. [Invention 1024] A polypeptide according to the present invention 1023, wherein the linker has a length of 4 to 40 amino acids. [Invention 1025] A polypeptide of the present invention 1023, wherein the linker comprises at least four glycine residues and / or serine residues. [Invention 1026] Linker (GGGGS) n A polypeptide of any of the Invention 1023 to 1025, comprising, where n is 1, 2, 3, 4, 5, or 6. [Invention 1027] A polypeptide according to any of invention 1024 to 1026, wherein the linker contains GSTSGGGSGGGSGGGGSS (SEQ ID NO: 32). [Invention 1028] The linker is the amino acid sequence (EAAAK) n Contains or amino acid sequence (EAAAK) n A polypeptide of the present invention 1023 or 1024, comprising, where n is 1, 2, 3, 4, 5, or 6. [Invention 1029] A polypeptide according to any of invention 1024 to 1026, wherein the linker contains GGGGS. [Invention 1030] Linker (GGGGS) 4 A polypeptide according to any of the invention 1023 to 1026. [Invention 1031] A polypeptide according to any of the present invention 1001 to 1030, wherein VH is amino-proximal to VL. [Invention 1032] A polypeptide according to any of the present invention 1001 to 1030, wherein VH is carboxylated to VL. [Invention 1033] A polypeptide according to any of the invention 1001 to 1032, comprising an scFv and a cytoplasmic region including a transmembrane domain and a primary intracellular signaling domain. [Invention 1034] A polypeptide according to any one of the invention 1001 to 1032, comprising a single transmembrane domain and / or a single cytoplasmic region comprising a primary intracellular signaling domain. [Invention 1035] A polypeptide according to the present invention 1033 or 1034, comprising, in order from the amino proximal terminus to the carboxyl proximal terminus, an scFv, a transmembrane domain, and a cytoplasmic region containing a primary intracellular signaling domain. [Invention 1036] A polypeptide according to the present invention 1033 or 1034, wherein the CAR is single-specific. [Invention 1037] A polypeptide according to any one of the invention 1033 to 1036, wherein the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv. [Invention 1038] A polypeptide according to the present invention 1037, wherein the extracellular spacer is 8 to 1000 amino acids long. [Invention 1039] A polypeptide according to the present invention 1038, wherein the extracellular spacer is 8 to 500 amino acids long. [Invention 1040] A polypeptide according to the present invention 1039, wherein the extracellular spacer is 100 to 300 amino acids long. [Invention 1041] A polypeptide according to the present invention 1039, wherein the extracellular spacer has fewer than 100 amino acids. [Invention 1042] A polypeptide according to any of invention 1037-1041, wherein the extracellular spacer is an IgG4 hinge, a CD8α hinge, an IgG1 hinge, a CD34 hinge, or a fragment thereof. [Invention 1043] Polypeptide of the present invention 1042, wherein the extracellular spacer comprises an IgG4 hinge or a fragment thereof. [Invention 1044] A polypeptide according to any of the inventions 1037 to 1043, wherein the extracellular spacer comprises a CH1 region, a CH2 region, and / or a CH3 region, or further comprises a CH3 region. [Invention 1045] Polypeptide of the present invention 1044, wherein the spacer comprises an IgG4 hinge polypeptide, a CH2 region, and a CH3 region, or consists of an IgG4 hinge polypeptide, a CH2 region, and a CH3 region. [Invention 1046] Polypeptides of the present invention 1044 or 1045, wherein the CH2 region includes L235E substitution and / or N297Q substitution. [Invention 1047] A polypeptide according to any one of invention 1042 to 1046, wherein the spacer comprises a polypeptide or a fragment thereof having at least 80% sequence identity with SEQ ID NO:36. [Invention 1048] A polypeptide according to any of the invention 1033 to 1047, wherein the transmembrane domain is the transmembrane domain of the α or β chain of a T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. [Invention 1049] Polypeptide 1048 of the present invention, wherein the transmembrane domain is a CD28 transmembrane domain. [Invention 1050] The polypeptide of the present invention 1048, wherein the transmembrane domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:37. [Invention 1051] A polypeptide according to any of the invention 1033 to 1050, wherein the primary intracellular signaling domain is CD3ζ. [Invention 1052] The polypeptide of the present invention 1051, wherein the primary intracellular signaling domain comprises a polypeptide or a fragment thereof having at least 80% sequence identity with SEQ ID NO:39. [Invention 1053] A polypeptide according to any of the invention 1033 to 1051, wherein the cytoplasmic region further comprises one or more costimulatory domains. [Invention 1054] A polypeptide according to any of the present invention 1033 to 1053, wherein the cytoplasmic region contains two costimulatory domains. [Invention 1055] Polypeptides of the present invention 1053 or 1054, wherein one or more co-stimulatory domains include co-stimulatory domains derived from one or more of 4-1BB(CD137), CD28, IL-15Rα, OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a / CD18), and / or ICOS(CD278). [Invention 1056] A polypeptide of the present invention 1055, wherein one or more co-stimulatory domains include a CD28-derived co-stimulatory domain. [Invention 1057] The polypeptide of the present invention 1056, wherein the co-stimulatory domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:38. [Invention 1058] A polypeptide according to any one of the present invention 1033 to 1057, further comprising a twisted linker between the transmembrane domain and the cytoplasmic region. [Invention 1059] A polypeptide according to the present invention 1058, wherein the twisted linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. [Invention 1060] A polypeptide according to the present invention 1059, wherein the amino acid residues include or consist of alanine residues. [Invention 1061] Polypeptide 1059 of the present invention, wherein the twisted linker consists of two or four alanine residues. [Invention 1062] A polypeptide comprising a CAR, wherein the CAR comprises, in order from the amino proximal terminus to the carboxyl proximal terminus, an scFv, a transmembrane domain, a twisted linker, and a cytoplasmic region containing a primary intracellular signaling domain, wherein the twisted linker contains 1 to 12 alanine residues. [Invention 1063] Polypeptide 1062 of the present invention, wherein the twisted linker contains or consists of two or four alanine residues. [Invention 1064] A polypeptide of the present invention 1062 or 1063, wherein scFv comprises VH and VL, and a linker is present between VH and VL. [Invention 1065] A polypeptide according to the present invention 1064, wherein the linker has a length of 4 to 40 amino acids. [Invention 1066] Polypeptide 1064 of the present invention, wherein the linker comprises at least four glycine residues and / or serine residues. [Invention 1067] Linker (GGGGS) n A polypeptide of any of the Invention 1064 to 1066, comprising, where n is 1, 2, 3, 4, 5, or 6. [Invention 1068] A polypeptide according to any of invention 1065 to 1066, wherein the linker contains GSTSGGGSGGGSGGGGSS (SEQ ID NO: 32). [Invention 1069] The linker is the amino acid sequence (EAAAK) n Contains or amino acid sequence (EAAAK) n A polypeptide of the present invention 1064 or 1065, comprising, where n is 1, 2, 3, 4, 5, or 6. [Invention 1070] A polypeptide according to any of invention 1065 to 1067, wherein the linker contains GGGGS. [Invention 1071] Linker (GGGGS) 4 A polypeptide according to any of the invention 1064 to 1067. [Invention 1072] A polypeptide according to any of the invention 1062 to 1071, wherein VH is amino-proximal to VL. [Invention 1073] A polypeptide according to any of the invention 1062 to 1071, wherein VH is carboxylated to VL. [Invention 1074] A polypeptide according to any one of the invention 1062 to 1073, comprising a single transmembrane domain and / or a single cytoplasmic region comprising a primary intracellular signaling domain. [Invention 1075] A polypeptide according to any of the present invention 1062 to 1074, wherein the CAR is single-specific. [Invention 1076] A polypeptide according to any of the present invention 1062 to 1074, wherein CAR is bispecific. [Invention 1077] A polypeptide according to any of invention 1062 to 1076, wherein the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv. [Invention 1078] Polypeptide 1077 of the present invention, wherein the extracellular spacer is 8 to 1000 amino acids long. [Invention 1079] Polypeptide 1078 of the present invention, wherein the extracellular spacer is 8 to 500 amino acid long. [Invention 1080] A polypeptide according to the present invention 1079, wherein the extracellular spacer is 100 to 300 amino acids long. [Invention 1081] A polypeptide according to the present invention 1079, wherein the extracellular spacer has fewer than 100 amino acids. [Invention 1082] Polypeptides of any of invention 1077-1081, wherein the extracellular spacer is an IgG4 hinge, a CD8α hinge, an IgG1 hinge, a CD34 hinge, or a fragment thereof. [Invention 1083] Polypeptide of the present invention 1082, wherein the extracellular spacer comprises an IgG4 hinge or a fragment thereof. [Invention 1084] A polypeptide according to any of the Invention 1077-1083, wherein the extracellular spacer comprises a CH1 region, a CH2 region, and / or a CH3 region, or further comprises a CH3 region. [Invention 1085] Polypeptide of the present invention 1084, wherein the spacer comprises an IgG4 hinge polypeptide, a CH2 region, and a CH3 region, or consists of an IgG4 hinge polypeptide, a CH2 region, and a CH3 region. [Invention 1086] Polypeptides of the present invention 1084 or 1085, comprising L235E substitution and / or N297Q substitution in the CH2 region. [Invention 1087] A polypeptide according to any one of Invention 1082 to 1086, wherein the spacer comprises a polypeptide or a fragment thereof having at least 80% sequence identity with SEQ ID NO:36. [Invention 1088] A polypeptide according to any of the inventions 1062 to 1087, wherein the transmembrane domain is the transmembrane domain of the α or β chain of a T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. [Invention 1089] Polypeptide 1088 of the present invention, wherein the transmembrane domain is a CD28 transmembrane domain. [Invention 1090] The polypeptide of the present invention 1089, wherein the transmembrane domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:37. [Invention 1091] A polypeptide according to any of the invention items 1062 to 1090, wherein the primary intracellular signaling domain is CD3ζ. [Invention 1092] The polypeptide of the present invention 1091, wherein the primary intracellular signaling domain comprises a polypeptide or a fragment thereof having at least 80% sequence identity with SEQ ID NO:39. [Invention 1093] A polypeptide according to any of the inventions 1062 to 1092, wherein the cytoplasmic region further comprises one or more costimulatory domains. [Invention 1094] A polypeptide according to any of invention 1062 to 1093, wherein the cytoplasmic region contains two costimulatory domains. [Invention 1095] Polypeptides of the present invention 1093 or 1094, wherein one or more co-stimulatory domains include one or more co-stimulatory domains derived from one or more of 4-1BB(CD137), CD28, IL-15Rα, OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a / CD18), and / or ICOS(CD278). [Invention 1096] A polypeptide of the present invention 1095, wherein one or more co-stimulatory domains include a CD28-derived co-stimulatory domain. [Invention 1097] The polypeptide of the present invention 1096, wherein the co-stimulatory domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:38. [Invention 1098] Polypeptides of any of Invention 1062 to 1097, wherein the scFv is a hybrid scFv comprising a framework region (FR) and a complementarity-determining region (CDR) derived from different antigen-binding regions or antibodies that bind to the same antigen. [Invention 1099] A polypeptide of the present invention 1098, wherein FR and CDR are derived from an antigen-binding region or antibody of the same species. [Invention 1100] Polypeptides of the present invention 1098 or 1099, wherein FR and CDR are derived from a human antigen-binding region or antibody. [Invention 1101] The polypeptide comprising a hybrid scFv is no significantly different from the polypeptide comprising a non-hybrid scFv, wherein the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, or the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv, wherein the polypeptide comprises any of the present inventions 1098 to 1100. [Invention 1102] A polypeptide according to any of the Invention 1098-1101, wherein FR and CDR are derived from an antigen-binding region or antibody having different tonic signaling intensities. [Invention 1103] Polypeptide of the present invention 1102, wherein FR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which CDR is derived. [Invention 1104] The polypeptide of the present invention 1102, wherein the CDR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which the FR originates. [Invention 1105] The polypeptide according to any of the Invention 1098 to 1104, wherein the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1106] The polypeptide according to any of the Invention 1098 to 1105, wherein the polypeptide has increased tumor cell clearance activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1107] A polypeptide according to any of the inventions 1062 to 1106, wherein the scFv contains anti-CD20 scFv. [Invention 1108] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1107, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29, 30, 31, 22, 12, 13, and 14; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, 27, 28, 18, 9, 10, and 11. [Invention 1109] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1107, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 5; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 6. [Invention 1110] The polypeptide of Invention 1107, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 24, 13, and 25, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, 10, and 23, respectively. [Invention 1111] The polypeptide of the present invention 1107, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:3, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:4. [Invention 1112] scFv Light chain variable regions including LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4, in order from the amino proximal end to the carboxyl proximal end; and The heavy chain variable region includes HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4, in order from the amino proximal end to the carboxyl proximal end of the heavy chain variable region. The polypeptide of the present invention 1107, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 15, 16, 17, 18, 9, 10, and 23. [Invention 1113] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1107, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:7; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:8. [Invention 1114] The polypeptide of the present invention 1107, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 12, 13, and 14, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, 10, and 11, respectively. [Invention 1115] The polypeptide of the present invention 1107, wherein scFv comprises VL including LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:1, and VH including HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:2. [Invention 1116] A polypeptide according to any of the inventions 1062 to 1097, wherein the scFv contains anti-GD2 scFv. [Invention 1117] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1116, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 136, 137, 138, 139, 113, 114, and 115; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 132, 133, 134, 135, 110, 111, and 112. [Invention 1118] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1116, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 141; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 140. [Invention 1119] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1116, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 120, 121, 122, 123, 129, 130, and 131; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with SEQ ID NO: 116, 117, 118, 119, 126, 127, and 128. [Invention 1120] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1116, comprising, where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain amino acid sequences having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 144; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain amino acid sequences having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 143. [Invention 1121] The polypeptide of the present invention 1116, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 129, 130, and 131, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 126, 127, and 128, respectively. [Invention 1122] The polypeptide of the present invention 1116, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:125, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:124. [Invention 1123] The polypeptide of the present invention 1116, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 113, 114, and 115, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 110, 111, and 112, respectively. [Invention 1124] The polypeptide of the present invention 1116, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:109, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:108. [Invention 1125] A polypeptide comprising an scFv containing a variable heavy chain (VH) region and a variable light chain (VL) region in order from the amino proximal terminus to the carboxyl proximal terminus, and a CAR containing a transmembrane domain and a cytoplasmic region containing a primary intracellular signaling domain, wherein the scFv is a hybrid scFv containing a framework region (FR) and a complementarity-determining region (CDR) derived from different antigen-binding regions or antibodies that bind to the same antigen. [Invention 1126] Polypeptide of the present invention 1125, wherein FR and CDR are derived from an antigen-binding region or antibody of the same species. [Invention 1127] Polypeptides of the present invention 1125 or 1126, wherein FR and CDR are derived from human or humanized antigen-binding regions or antibodies. [Invention 1128] The polypeptide according to any of the Invention 1125 to 1127, wherein the immunogenicity of the polypeptide containing the hybrid scFv is not significantly different from that of the polypeptide containing the non-hybrid scFv, wherein the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, or the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1129] Polypeptides of any of the present invention 1125 to 1128, wherein FR and CDR are derived from antigen-binding regions or antibodies having different tonic signaling intensities. [Invention 1130] Polypeptide of the present invention 1129, wherein FR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which CDR is derived. [Invention 1131] The polypeptide of the present invention 1129, wherein the CDR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which the FR is derived. [Invention 1132] The polypeptide according to any of the Invention 1125 to 1131, wherein the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1133] The polypeptide according to any of the Invention 1125 to 1132, wherein the polypeptide has increased tumor cell clearance activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1134] The polypeptide according to any of the present invention 1125 to 1133, wherein the polypeptide comprises a twisted linker between a transmembrane domain and a cytoplasmic domain, and the twisted linker comprises 1 to 12 alanine residues. [Invention 1135] Polypeptide 1134 of the present invention, wherein the twisted linker contains or consists of two or four alanine residues. [Invention 1136] A polypeptide of the present invention 1134 or 1135, wherein scFv comprises VH and VL, and a linker is present between VH and VL. [Invention 1137] A polypeptide according to the present invention 1136, wherein the linker has a length of 4 to 40 amino acids. [Invention 1138] A polypeptide according to the present invention 1137, wherein the linker comprises at least four glycine residues and / or serine residues. [Invention 1139] Linker (GGGGS) n A polypeptide of any of the Invention 1136-1138, comprising, where n is 1, 2, 3, 4, 5, or 6. [Invention 1140] A polypeptide according to any of inventions 1136 to 1139, wherein the linker contains GSTSGGGSGGGSGGGGSS (SEQ ID NO: 32). [Invention 1141] A polypeptide according to any of the present inventions 1125 to 1140, wherein VH is amino-proximal to VL. [Invention 1142] A polypeptide according to any of the present inventions 1125 to 1140, wherein VH is carboxylated to VL. [Invention 1143] A polypeptide according to any of invention 1125 to 1142, wherein the CAR is single-specific. [Invention 1144] A polypeptide according to any of invention 1125 to 1142, wherein CAR is bispecific. [Invention 1145] A polypeptide according to any of the present invention 1125 to 1144, wherein the CAR further comprises an extracellular spacer between the transmembrane domain and the scFv. [Invention 1146] A polypeptide according to the present invention 1145, wherein the extracellular spacer is 8 to 1000 amino acids long. [Invention 1147] A polypeptide according to the present invention 1145, wherein the extracellular spacer is 8 to 500 amino acids long. [Invention 1148] A polypeptide according to the present invention 1145, wherein the extracellular spacer is 100 to 300 amino acids long. [Invention 1149] A polypeptide according to the present invention 1145, wherein the extracellular spacer has fewer than 100 amino acids. [Invention 1150] Polypeptides of any of Invention 1145-1149, wherein the extracellular spacer is an IgG4 hinge, a CD8α hinge, an IgG1 hinge, a CD34 hinge, or a fragment thereof. [Invention 1151] Polypeptide of the present invention 1150, wherein the extracellular spacer comprises an IgG4 hinge or a fragment thereof. [Invention 1152] A polypeptide according to any of the Invention 1145-1152, wherein the extracellular spacer comprises a CH1 region, a CH2 region, and / or a CH3 region, or further comprises a CH3 region. [Invention 1153] The polypeptide of the present invention 1152, wherein the spacer comprises an IgG4 hinge polypeptide, a CH2 region, and a CH3 region, or consists of an IgG4 hinge polypeptide, a CH2 region, and a CH3 region. [Invention 1154] A polypeptide of the present invention 1152 or 1153, comprising an L235E substitution and / or an N297Q substitution in the CH2 region. [Invention 1155] The spacer comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:36, any polypeptide according to Invention 1154. [Invention 1156] A polypeptide according to any of the invention items 1125 to 1155, wherein the transmembrane domain is the transmembrane domain of the α or β chain of a T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154. [Invention 1157] A polypeptide according to the present invention 1156, wherein the transmembrane domain is a CD28 transmembrane domain. [Invention 1158] The polypeptide of the present invention 1157, wherein the transmembrane domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:37. [Invention 1159] A polypeptide according to any of the invention items 1125 to 1158, wherein the primary intracellular signaling domain is CD3ζ. [Invention 1160] The polypeptide of the present invention 1159, wherein the primary intracellular signaling domain comprises a polypeptide or a fragment thereof having at least 80% sequence identity with SEQ ID NO:39. [Invention 1161] A polypeptide according to any of the present inventions 1125 to 1160, wherein the cytoplasmic region further comprises one or more costimulatory domains. [Invention 1162] A polypeptide according to any of invention 1125 to 1161, wherein the cytoplasmic region contains two costimulatory domains. [Invention 1163] A polypeptide according to Invention 1161 or 1162, wherein one or more co-stimulatory domains include one or more co-stimulatory domains derived from one or more of 4-1BB(CD137), CD28, IL-15Rα, OX40, CD2, CD27, CDS, ICAM-1, LFA-1(CD11a / CD18), and / or ICOS(CD278). [Invention 1164] A polypeptide according to any one of the invention 1161 to 1163, wherein one or more co-stimulatory domains include a CD28-derived co-stimulatory domain. [Invention 1165] The polypeptide of the present invention 1164, wherein the co-stimulatory domain comprises a polypeptide or fragment thereof having at least 80% sequence identity with SEQ ID NO:38. [Invention 1166] A polypeptide according to any of invention 1125 to 1165, wherein scFv contains anti-CD20 scFv. [Invention 1167] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1166, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29, 30, 31, 22, 12, 13, and 14; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, 27, 28, 18, 9, 10, and 11. [Invention 1168] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1166, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 5; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 6. [Invention 1169] The polypeptide of Invention 1166, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 24, 13, and 25, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, 10, and 23, respectively. [Invention 1170] The polypeptide of Invention 1166, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:3, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:4. [Invention 1171] scFv Light chain variable regions including LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, and LFR4, in order from the amino proximal end to the carboxyl proximal end; and The heavy chain variable region includes HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, and HFR4, in order from the amino proximal end to the carboxyl proximal end of the heavy chain variable region. The polypeptide of the present invention 1166, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19, 20, 21, 22, 24, 13, and 25; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15, 16, 17, 18, 9, 10, and 23. [Invention 1172] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1166, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:7; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:8. [Invention 1173] The polypeptide of the present invention 1166, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 12, 13, and 14, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, 10, and 11, respectively. [Invention 1174] The polypeptide of the present invention 1166, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:1, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:2. [Invention 1175] A polypeptide according to any of invention 1125 to 1165, wherein the scFv contains anti-GD2 scFv. [Invention 1176] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1175, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 136, 137, 138, 139, 113, 114, and 115; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 132, 133, 134, 135, 110, 111, and 112. [Invention 1177] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1175, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 141; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 140. [Invention 1178] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1175, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 120, 121, 122, 123, 129, 130, and 131; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 116, 117, 118, 119, 126, 127, and 128. [Invention 1179] scFv The light chain variable region (VL) includes, in order from the amino proximal end to the carboxyl proximal end, the light chain framework region 1 (LFR1), the light chain complementarity determination region 1 (LCDR1), the light chain framework region 2 (LFR2), the light chain complementarity determination region 2 (LCDR2), the light chain framework region 3 (LFR3), the light chain complementarity determination region 3 (LCDR3), and the light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). The polypeptide of the present invention 1175, comprising; where LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence having at least 90% sequence identity with LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO: 144; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each comprise an amino acid sequence having at least 90% sequence identity with HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO: 143. [Invention 1180] The polypeptide of Invention 1175, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 129, 130, and 131, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 126, 127, and 128, respectively. [Invention 1181] The polypeptide of the present invention 1107, wherein scFv comprises VL, which includes LCDR1, LCDR2, and LCDR3 of the variable region of SEQ ID NO:125, and VH, which includes HCDR1, HCDR2, and HCDR3 of the variable region of SEQ ID NO:124. [Invention 1182] The polypeptide of the present invention 1175, wherein scFv comprises VL containing LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 113, 114, and 115, respectively; and VH containing HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 110, 111, and 112, respectively. [Invention 1183] The polypeptide of Invention 1175, wherein scFv comprises VL, which includes the variable region LCDR1, LCDR2, and LCDR3 of SEQ ID NO:109, and VH, which includes the variable region HCDR1, HCDR2, and HCDR3 of SEQ ID NO:108. [Invention 1184] A nucleic acid comprising a sequence encoding any polypeptide according to invention 1001 to 1183. [Invention 1185] The nucleic acid of the present invention 1184, which is an expression construct. [Invention 1186] The nucleic acid of the present invention 1185, wherein the expression construct is a viral vector. [Invention 1187] The nucleic acid of the present invention 1186, wherein the viral vector includes a retroviral vector and a retrovirus-derived vector. [Invention 1188] The nucleic acid of the present invention 1187, wherein the viral vector is a lentiviral vector or a vector derived from a lentivirus. [Invention 1189] A lentiviral vector comprising a sequence encoding any polypeptide according to invention 1001 to 1183. [Invention 1190] A cell containing any nucleic acid according to Invention 1184 to 1189. [Invention 1191] A cell containing the nucleic acid of the present invention 1190, in which a viral vector is incorporated into the cell's genome. [Invention 1192] A cell expressing any polypeptide according to invention 1001 to 1183. [Invention 1193] Cells according to any of the invention 1190 to 1192, which are T cells, natural killer (NK) cells, natural killer T cells (NKT), invariant natural killer T cells (iNKT), stem cells, lymphocyte progenitor cells, peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal hepatocytes, embryonic stem cells, hematopoietic stem cells or hematopoietic progenitor cells (HSPCs), umbilical cord blood cells, or induced pluripotent stem cells (iPS cells). [Invention 1194] The cells of the present invention 1193, which are T cells or NK cells. [Invention 1195] Cells according to Invention 1194, in which T cells include naive memory T cells. [Invention 1196] The cells of the present invention 1195, wherein the naive memory T cells include CD4+ T cells or CD8+ T cells. [Invention 1197] A cell population containing any of the cells described in Invention 1190 to 1196. [Invention 1198] 10 3 ~10 8 A cell population according to the present invention 1197, containing individual cells. [Invention 1199] A composition comprising a polypeptide according to any of Invention 1001 to 1183, a nucleic acid according to any of Invention 1184 to 1189, a cell according to any of Invention 1190 to 1196, or a cell population according to Invention 1197 or 1198. [Invention 1200] A method for producing cells that express a polypeptide, comprising the step of introducing any nucleic acid according to Invention 1184 to 1189 into cells. [Invention 1201] A method for producing a CAR, comprising the step of introducing a twisted linker between the transmembrane domain and the cytoplasmic domain of the CAR, wherein the twisted linker comprises 1 to 12 alanine residues. [Invention 1202] A method for producing a CAR having a hybrid scFv and a cytoplasmic region including a transmembrane domain and a primary intracellular signaling domain, comprising the step of combining a CDR derived from a first antigen-binding region or antibody with a FR derived from a second antigen-binding region or antibody, wherein the first and second antigen-binding regions or antibodies bind to the same antigen. [Invention 1203] The method of the present invention 1202, wherein FR and CDR are derived from antigen-binding regions or antibodies of the same species. [Invention 1204] The method of the present invention 1202 or 1203, wherein FR and CDR are derived from a human antigen-binding region or antibody. [Invention 1205] A method according to any one of the present invention 1202 to 1204, wherein FR and CDR are derived from antigen-binding regions or antibodies having different tonic signaling intensities. [Invention 1206] The method of the present invention 1205, wherein FR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which CDR is derived. [Invention 1207] The method of the present invention 1205, wherein the CDR is derived from an antigen-binding region or antibody having a higher tonic signaling intensity than the antigen-binding region or antibody from which the FR originates. [Invention 1208] The method according to any one of the present invention 1202 to 1207, wherein the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1209] The method according to any one of the present invention 1202 to 1208, wherein the polypeptide has increased tumor cell clearance activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the FR of the hybrid scFv, and / or the polypeptide has increased tumor-killing activity compared to a CAR containing a non-hybrid scFv, where the non-hybrid scFv is derived from the same antigen-binding region or antibody as the CDR of the hybrid scFv. [Invention 1210] A method according to any one of the present invention 1200 to 1209, wherein a virus encoding the polypeptide is used to infect a cell. [Invention 1211] The method of the present invention 1210, wherein the virus comprises a lentivirus, or a virus or vector derived from a lentivirus. [Invention 1212] A method according to any one of the invention 1200 to 1211, wherein the cells are T cells, natural killer (NK) cells, natural killer T cells (NKT), invariant natural killer T cells (iNKT), stem cells, lymphocyte progenitor cells, peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal hepatocytes, embryonic stem cells, umbilical cord blood cells, or induced pluripotent stem cells (iPS cells). [Invention 1213] The method of the present invention 1212, wherein the cells are T cells or NK cells. [Invention 1214] The method of the present invention 1213, wherein the T cells include naive memory T cells. [Invention 1215] The method of the present invention 1214, wherein the naive memory T cells include CD4+ T cells or CD8+ T cells. [Invention 1216] Any method of the present invention 1213 to 1215, further comprising the step of culturing cells that are not yet T cells or NK cells under conditions that promote differentiation of the cells into T cells or NK cells. [Invention 1217] Any method of the present invention 1200 to 1216, further comprising the step of culturing the cells under conditions that increase the size of the cells, before and / or after introducing nucleic acids into the cells. [Invention 1218] The method of the present invention 1217, wherein cells are cultured in a serum-free medium. [Invention 1219] Cells prepared by any of the methods described in Invention 1200 or 1203-1218. [Invention 1220] A CAR manufactured by any of the methods described in invention 1201 to 1218. [Invention 1221] A step to provide a CAR having a hybrid scFv and a cytoplasmic region including a transmembrane domain and a primary intracellular signaling domain, comprising combining a CDR derived from a first antigen-binding region or antibody with a FR derived from a second antigen-binding region or antibody, wherein the first and second antigen-binding regions or antibodies bind to the same antigen; and Steps to evaluate the function of the CAR A method for screening CARs, including [specific data / information]. [Invention 1222] The method of the present invention 1221, wherein the step of evaluating the function of the CAR includes evaluating the tonic signaling intensity of the CAR. [Invention 1223] The method of the present invention 1222, comprising evaluating tonic signaling intensity by CDV dilution assay and / or expression evaluation by antibody staining for an activation marker. [Invention 1224] A method according to any one of the present invention 1221 to 1223, wherein the step of evaluating the function of CAR includes determining one or more of the following: tumor killing efficacy, tumor clearance efficacy, in vivo survival rate, in vivo tumor clearance, and in vivo tumor load reduction. [Invention 1225] The method of any of the present invention 1221 to 1224 further comprises the step of producing a CAR containing a hybrid scFv, which includes combining a CDR derived from a first antigen-binding region or antibody with a FR derived from a second antigen-binding region or antibody, wherein the first and second antigen-binding regions or antibodies bind to the same antigen. [Invention 1226] A method for treating a patient with cancer, comprising the step of administering an effective amount of the composition of the present invention 1199 to the patient. [Invention 1227] A method according to the present invention 1226 for treating cancer in patients with cancer. [Invention 1228] The method of the present invention 1226 or 1227, further comprising the step of administering an additional treatment to the patient. [Invention 1229] The method of the present invention 1228, wherein the additional treatment method includes immunotherapy. [Invention 1230] A method according to any one of the present invention 1226 to 1229, wherein the cancer includes lymphoma or neuroblastoma. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various variations and modifications that fall within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are provided only as illustrations, while illustrating specific aspects of the present invention. [Brief explanation of the drawing]

[0065] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0066] [Figure 1] Figures 1A-B. (A) Schematic diagrams of CD20 CARs constructed using scFv derived from various antibodies. All CARs were second-generation receptors containing a CD28 costimulatory domain. (B) Sequences of scFv domains used in CD20 CAR construction. [Figure 2] Figures 2A-B. Rituximab-derived CD20 CARs exhibit antigen-independent CAR signaling, resulting in (A) upregulation of activation and depletion markers and (B) T cell proliferation in the absence of antigen stimulation. CD19 CARs and GD2 CARs were included as negative and positive controls, respectively, for tonic signaling. [Figure 3] Figure 3. Schematic diagram of alanine insertion to induce structural rotation of the CAR molecule. [Figure 4] Figure 4. Alanine insertion calibrates CAR tonic signaling intensity. (A) T cell proliferation quantified by CellTrace Violoet dye dilution and (B) cytokine production quantified by ELISA in the absence of antigen stimulation demonstrate the varying degree of antigen-independent activation in T cells expressing rituximab-derived CD20 CARs containing varying numbers of alanine residues inserted between the transmembrane domain and the cytoplasmic CD28 domain. [Figure 5]Figure 5. Rituximab-derived CD20 CARs containing two alanine residues inserted between the transmembrane CD28 domain and the cytoplasmic CD28 domain exhibit superior in vivo antitumor activity. 500,000 firefly luciferase-expressing Raji lymphoma cells were engrafted in NOD / scid / γ- / -(NSG) mice and treated with 1.35 million T cells expressing either a CD20 CAR or a transduction marker (EGFRt) 7 days after tumor injection. 1.5 million T cells were re-administered to the animals 7 days later. All CARs tested in this study contained rituximab-derived scFv, an IgG4 hinge-CH2-CH3 extracellular spacer, the CD28 transmembrane domain and the CD28 cytoplasmic domain, and the CD3ζ signaling domain. 0–4 alanine residues were inserted between the CD28 transmembrane domain and the CD28 cytoplasmic domain. Tumor progression was monitored by bioluminescence imaging, and the radiated signal was shown as a function of time after tumor injection. The 2-alanine insertion variant showed superior tumor control and extended median survival compared to the parent (non-alanine insertion) construct and to other variants. [Figure 6] Figures 6A-B. (A) Schematic diagrams of CD20 CARs containing a Leu16-derived scFv domain, a rituximab-derived scFv domain, or a hybrid scFv domain. Two hybrids were constructed. Only the RFR-LCDR hybrid was functional (see Figure 7). All CARs were second-generation receptors containing a CD28 costimulatory domain, as shown in Figure 3. (B) Sequences of scFv domains used in CD20 CAR construction. [Figure 7] Figures 7A-B. The RFR-LCDR hybrid CD20 CAR exhibits antigen-independent CAR signaling, resulting in (A) upregulation of activation and depletion markers and (B) T cell proliferation in the absence of antigen stimulation. CD19 CAR and GD2 CAR were included as negative and positive controls, respectively, for tonic signaling. [Figure 8]Figure 8. The RFR-LCDR hybrid CD20 CAR exhibits superior tumor cell death in vitro during repeated antigen challenge. T cells expressing various CARs or transduction markers (EGFRt) were challenged with fresh Raji lymphoma cells every 48 hours. The number of viable target cells was quantified by flow cytometry before each rechallenge. Results from multiple donors demonstrate that tumor cell death with the RFR-LCDR hybrid CAR is superior to that of either the parent construct (Leu16 and rituximab). [Figure 9A] Figures 9A-C. The RFR-LCDR hybrid CD20 CAR exhibits superior tumor clearance in vivo. NSG mice were engrafted with 500,000 firefly luciferase-expressing Raji lymphoma cells and treated with two T cell doses as described in Figure 2. 55 days after the first T cell treatment, the animals were re-challenged with a second dose of tumor cells. (A) Tumor progression was monitored by bioluminescence imaging. (B) Survival curves are shown as Kaplan-Meier curves. The results show that the hybrid CAR is superior to both the parental CD20 CAR and CD19 CAR in tumor clearance and long-term recurrence prevention. (C) The frequency of CAR-expressing human T cells in peripheral blood 23 days after the first T cell injection was measured by flow cytometry. The addition of two alanine residues to the CAR improved the rituximab-based receptor, thus confirming the previous data shown in Figure 5. Combining hybrid CARs with two alanine insertions further accelerated the rate of early tumor clearance and resulted in a significant increase in T cell persistence compared to hybrid CARs without alanine insertions. This suggests that combining scFv modification and alanine insertion strategies can lead to synergistic improvements in CAR function. [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 10-1]Figure 10A-F. (A) Schematic diagram of a panel of second-generation anti-CD20 CARs, consisting of scFv derived from four different monoclonal antibodies fused with the IgG4 spacer, CD28 transmembrane domain and CD28 cytoplasmic domain, and CD3ζ signaling domain. The CARs were further fused with truncated EGFR (EGFRt), used as a transduction marker, via a self-cleaved T2A peptide (top). KD values ​​and CDR structural family designations of the four antibodies from which the scFv were derived (bottom). The sequences and structural family designations of FR and CDR were determined as previously described (Chothia and Lesk, 1987; Chothia et al., 1989; Kabat and Wu, 1971; Martin and Thornton, 1996). (B-C) NSG mice were intravenously (iv) injected with 0.5 × 10⁶ firefly luciferase-expressing Raji cells and treated 6 days later with iv delivery of 5 × 10⁶ CD20-targeted CAR-T cells. (B) Tumor progression was monitored by bioluminescence imaging (n=6 mice per group). The minimum and maximum values ​​on the radiance scale are 5 × 10⁴ and 1 × 10⁷, respectively. (C) Mean radiance (p / sec / cm² / sr) of individual animals for each test group. (D) Expression of activation and depletion markers on CAR+ T cells was evaluated 11 days after DynaBeads removal without CD20 antigen stimulation. Data bars show the mean ± 1 standard deviation (SD) of three technical replicates. Results are representative of three independent experiments using T cells from three different healthy donors. Unless otherwise noted, p-values ​​were determined by unpaired two-sided two-sample Student's t-test. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (E) 4-day T cell proliferation assay for CAR+ T cells with CellTrace Violet (CTV) dye in the absence or presence of target cells (on-target, CD19+ / CD20+ K562 cells; off-target, parental K562 cells) at an effector-to-target (E:T) ratio of 2:1. The data shown are representative of five independent experiments from five different healthy donors.(F) Metabolic rate of CD20 CAR-T cells cultured for 24 hours in RPMI supplemented with 10% dFBS and exogenous IL-2 and IL-15, without CD20 antigen stimulation. Data bars show the mean ± 1 SD of three technical replicates. Results are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 10-2] See the explanation in Figure 10-1. [Figure 11-1] Figures 11A-E. Twisted reorientation of signaling domains modulates CAR-T cell activity. (A) Schematic diagram of alanine incorporation into rituximab-based CARs. (B) 4-day T cell proliferation assay with CellTrace Violet (CTV) dye in the absence or presence of target cells at an E:T ratio of 2:1. The data shown are representative of three independent experiments from three different healthy donors. (C) TNFα production of CAR-T cells was measured 7 days after DynaBeads removal without CD20 antigen stimulation. The results are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (D, E) NSG mice were intravenously injected with 0.5 × 10⁶ firefly luciferase-expressing Raji cells, followed by two doses of CAR+ T cells at 6 days (1.35 × 10⁶ cells) and 12 days (1.5 × 10⁶ cells). Each group consisted of n=6 mice. (D) Tumor progression was monitored by bioluminescence imaging (top). The mean radiance (p / sec / cm² / sr) of individual animals is shown for each group (bottom). (E) Kaplan-Meier survival curves. Statistical significance was determined by the log-rank (Mantel-Cox) test. *p<0.05, **p 0.01, ***p<0.001, ns not statistically significant. [Figure 11-2] See the explanation in Figure 11-1. [Figure 12A]Figures 12A-E. scFv sequence hybridization produces functionally superior CAR variants. (A) Schematic diagram of scFv sequence hybridization in CAR molecules. The framework region (FR) and complementarity-determining region (CDR) of scFv derived from Leu16 and rituximab were mixed together to produce two novel CAR variants. (B) RFR-LCDR hybrid CAR-T cells exhibit superior antitumor function and T cell proliferation upon repeated antigen challenge. CAR-T cells were challenged with Raji cells (CD19+ / CD20+) every two days at an E:T ratio of 2:1. The number of T cells and target cells was quantified by flow cytometry. The data shown are mean ± 1 SD of three technical replicates. Results are representative of three independent experiments from three different healthy donors. (C) Expression of activation and depletion markers was assessed 11 days after DynaBeads removal without CD20 antigen stimulation. Data bars show mean ± 1 SD of three technical replicates. The results are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (D) 4-day CAR-T cell proliferation assay with CellTrace Violet (CTV) dye in the absence or presence of target cells at an E:T ratio of 2:1. (E) Metabolic analysis of CAR-T cells in culture in the absence of antigen stimulation. CAR-T cells were cultured for 72 hours in RPMI supplemented with 10% thermoinactivated dialysis fetal bovine serum (HI-dFBS), IL-2, and IL-15. Data bars show the mean ± 1 SD of three technical replicates. The data are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 12B] See the explanation in Figure 12A. [Figure 12C] See the explanation in Figure 12A. [Figure 12D] See the explanation in Figure 12A. [Figure 12E] See the explanation in Figure 12A. [Figure 13-1]Figures 13A-G. scFv hybridization combined with twist reorientation in CAR proteins further enhances CAR-T cell function in vivo. (A-D) NSG mice were intravenously injected with firefly luciferase-expressing Raji cells, followed by two doses of CAR+ T cells and one Raji tumor rechallenge. (A) Schematic diagram of the in vivo experiment (n=6 mice per group). (B) Tumor signal in individual animals quantified by bioluminescence imaging. (C) Kaplan-Meier survival curves. Log-rank (Mantel-Cox) tests were performed for pairwise comparisons. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (D) Frequency of human CD45+EGFRt+ cells in peripheral blood collected from mice 23 days after the first T cell injection. (E-G) NSG mice were intravenously injected with firefly luciferase-expressing Raji cells, followed by one dose of CAR+ T cells. Mice were rechallenged twice with Raji cells on days 25 and 45 after T cell injection. (E) Schematic diagram of the in vivo experiment (n=6 mice per group). (F) Tumor signal in individual animals quantified by bioluminescence imaging. (G) Frequency of human CD45+EGFRt+ cells in peripheral blood collected from mice over time. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 13-2] See the explanation in Figure 13-1. [Figure 13-3] See the explanation in Figure 13-1. [Figure 14A]Figures 14A-C. Transcriptome and epigenetic analysis revealed CAR-dependent variability in the T cell phenotype. (A, B) NSG mice were intravenously injected with 0.5 × 10⁶ firefly luciferase-expressing Raji cells and treated 6 days later with intravenous delivery of 2.85 × 10⁶ CAR+ T cells. Nine days after T cell injection, liver, spleen, cardiac blood, and bone marrow were collected from tumor-carrying mice (n=2 mice per group). CAR+ T cells were obtained by enriching the huCD45+EGFRt+ population and subsequently analyzed by RNA-seq and ATAC-seq. (A) Volcano plot of differentially expressed genes in comparison between rituximab CAR-T cells and Leu16 CAR-T cells (left), RFR-LCDR CAR-T cells (center), or RFR-LCDR.AA CAR-T cells (right), based on RNA-seq. All differentially expressed genes are plotted in gray. Genes with FDR < 0.05 and at least 2x upregulation or downregulation (log2FC > 1 or log2FC < -1) are shown as red dots. Genes that appear in all three pairs of comparisons with log2FC > 1 and FDR < 0.05 or log2FC < -1 and FDR < 0.05 are marked. (B) Genome browser files of differentially accessible regions at the ATP9A locus, KIR2DL3 locus, KIR2DL1 locus, and KIR3DL1 locus of Leu16 CAR-T cells, rituximab CAR-T cells, RFR-LCDR CAR-T cells, and RFR-LCDR.AA CAR-T cells. (C) Animals treated with rituximab-based CAR-T cells show reduced serum glucose levels. Serum was collected from animals 58 days after T-cell injection in the studies shown in Figures 13E–G. Serum glucose concentrations were measured by LC-MS. Data bars show the mean ± 1 SD of the biological replicates (n=6 for both hybrid CAR-T cell groups; n=4 for the rituximab.AA CAR-T cell treatment group due to death caused by tumor volume prior to the day of sample collection).*p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 14B] See the explanation in Figure 14A. [Figure 14C] See the explanation in Figure 14A. [Figure 15A] Figures 15A-D. RFR-LCDR.AA CAR-T cells exhibit robust T cell activation associated with the memory phenotype. (A) Gene set enrichment analysis (GSEA) was performed on RNA-seq data obtained as described in Figure 14A. A summary of the results in pathways related to T cell phenotype and function is shown in BubbleGUM map format (Spinelli et al., 2015). (B) Mountain plot of pathways related to T cell subtype. (C) Mountain plot of pathways related to T cell activation and interferon-γ signaling. (D) Mountain plot of pathways related to the cell cycle, DNA replication, and cellular metabolism. [Figure 15B] See the explanation in Figure 15A. [Figure 15C] See the explanation in Figure 15A. [Figure 15D] See the explanation in Figure 15A. [Figure 16-1]Figures 16A-D. Panel showing whether CD20 CARs exhibit similar features in vitro. (A) Alignment of scFv sequences of leu16, rituximab, GA101, and ofatumumab using T-Coffee (Notredame et al., 2000). (B) CAR-T cell transduction efficiency quantified by CAR surface expression (top) and transduction marker expression (bottom), detected via antibody staining of IgG4 extracellular spacer (Fc) and EGFRt of the CAR, respectively. Median fluorescence intensity (MFI) and % positivity for each antigen stain are noted below the flow cytometry histogram. Results are representative of three independent experiments from three different healthy donors. (C) CD20 CAR-T cell proliferation in ex vivo culture with exogenous cytokines IL-2 and IL-15. Fold changes in total viable cell count between day 2 and day 14 are shown. Each data point represents one donor; for each construct, data for four donors are shown. No statistically significant differences were detected between any of the donors by an unpaired two-sided two-sample Student's t-test. (D) Cytotoxicity and proliferation of CAR-T cells during repeated antigen challenge. CD20 CAR-T cells were challenged with Raji tumor cells every two days at an effector-to-target (E:T) ratio of 2:1, and the number of viable Raji cells and CAR-T cells was quantified by flow cytometry. The data shown are mean values ​​of three technical replicates with error bars indicating ±1 standard deviation (SD). Results are representative of three independent experiments from three different healthy donors. [Figure 16-2] See the explanation in Figure 16-1. [Figure 17-1]Figures 17A-E. Tumor progression and postmortem CD20 CAR-T cell characterization in a Raji xenograft model. (A) Firefly luciferase-expressing Raji cells were engrafted in NSG mice and treated with CD20 CAR-T cells as shown in Figure 10B. Tumor growth rate between 12 days post-T cell injection and the humane endpoint was quantified by measuring tumor volume by bioluminescence imaging and fitting exponential regression curves to the tumor progression data of each individual animal. The mean doubling time (T doubling) of tumor signal for each group of mice (n=6) is shown above each plot. (B, C) Frequency of tumor cells (B) and human CD45+EGFRt+ cells (B) in bone marrow, brain, liver, and spleen collected from mice at the time of euthanasia, quantified by flow cytometry. (D, E) Frequency of PD-1+ cells (D) and LAG-3+ cells (E) among human CD45+EGFRt+ cells collected from mice at the time of euthanasia, quantified by flow cytometry. No statistically significant differences were detected between any of the donors by unpaired two-sided two-sample Student's t-test for all data shown in panels (B) to (E). [Figure 17-2] See the explanation in Figure 17-1. [Figure 17-3] See the explanation in Figure 17-1. [Figure 18] Figure 18. Rituximab-treated CAR-T cells are metabolically more active than other CD20 CAR-T cells. Uptake of amino acids and other nutrients by CAR-T cells cultured for 24 hours in RPMI supplemented with 10% thermoinactivated dialysis fetal bovine serum (HI-dFBS), IL-2, and IL-15. Data bars represent the mean ± 1 SD of three technical replicates. Results are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. Results in this figure are from the same experiment as Figure 10F. Each set of four bars, from left to right, represents data for EGFRt, CD19, Leu16, and rituximab, respectively. [Figure 19]Figure 19. CD20 CARs are uniformly distributed on the T cell surface in the absence of antigen engagement. Jurkat cells transduced by CAR-HaloTag fusion proteins were stained with the red fluorescent dye tetramethylrhodamine (TMR) and imaged using confocal microscopy. CAR molecules are uniformly distributed on the cell surface in the absence of antigen stimulation. [Figure 20-1] Figures 20A-D. Rituximab-based CAR-T cells with twisted reorientation exhibit similar in vitro cytotoxicity and antigen-independent activation marker expression. (A) Schematic diagram of rituximab-based CAR constructs with 0-4 alanines inserted between the CD28 transmembrane domain and cytoplasmic domain. (B) CAR surface expression was quantified by antibody staining of HA tags fused to the N-terminus of each CAR. Data are representative of three independent experiments from three different healthy donors. (C) Cytotoxicity and proliferation of CAR-T cells during repeated antigen challenge. CD20 CAR-T cells were challenged with Raji tumor cells every two days at an E:T ratio of 2:1, and the number of viable Raji cells and CAR-T cells was quantified by flow cytometry. Data shown are mean values ​​of three technical replicates with error bars indicating ±1 standard deviation (SD). Results are representative of three independent experiments from three different healthy donors. (D) Without CD20 antigen stimulation, the expression of activating and depleting markers was assessed 11 days after DynaBeads removal. Data bars show the mean ± 1 SD of three technically replicated trials. Results are representative of three independent experiments from three different healthy donors. Unless otherwise noted, p-values ​​were determined by unpaired two-tailed Student's t-tests. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 20-2] See the explanation in Figure 20-1. [Figure 21-1]Figures 21A-E. GD2 CAR-T cells with twisted reorientation exhibit differential tumor control in vivo despite the same in vitro performance. (A) Schematic diagram of GD2 CAR constructs with 0-4 alanine molecules inserted between the CD28 transmembrane domain and the cytoplasmic domain. (B) CAR surface expression (top) and transduction efficiency (bottom) were quantified by antibody staining of Fc and EGFRt. (C) Cytotoxicity and proliferation of CAR-T cells during repeated antigen challenge. GD2 CAR-T cells were challenged with CHLA-255 tumor cells every two days at an E:T ratio of 2:1, and the number of viable Raji cells and CAR-T cells was quantified by flow cytometry. The data shown are the mean of three technical replicates with error bars indicating ±1 standard deviation (SD). (D, E) NSG mice were intravenously injected with 3.5 × 10⁶ firefly luciferase-expressing CHLA-255 cells, and 17 days later, they were intravenously injected with 2 × 10⁶ GD2 CAR-T cells. (D) Tumor progression was monitored by bioluminescence imaging. (E) The frequency of human CD45+EGFRt+ cells in the liver and spleen collected from mice at the time of euthanasia was quantified by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 21-2] See the explanation in Figure 21-1. [Figure 22-1]Figure 22A-D. In vitro characterization of hybrid CAR-T cells. (A) CAR surface expression (top) and transduction efficiency (bottom) were quantified by antibody staining of Fc and EGFRt. Results are representative of three independent experiments from three different healthy donors. (B) CAR molecules are uniformly distributed on the T cell surface in the absence of antigen stimulation. Jurkat cells transduced with CAR-HaloTag fusion protein were stained with TMR and imaged by confocal microscopy. (C, D) Production of IFNγ, TNFα, and IL-2 in the presence (C) and absence (D) of CD19+ / CD20+ K562 target cells. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. For (C) and (D), each set of seven bars, from left to right, represents data for EGFRt, CD19, Leu16, rituximab, rituximab.AA, RFR-LCDR, and RFR-LCDR.AA, respectively. [Figure 22-2] See the explanation in Figure 22-1. [Figure 23] Figure 23. Hybrid CAR-T cells exhibit reduced metabolic activity compared to rituximab CAR-T cells. Uptake of amino acids and other nutrients by CAR-T cells cultured for 72 hours in RPMI supplemented with 10% thermoinactivated dialysis fetal bovine serum (HI-dFBS), IL-2, and IL-15. Data bars represent the mean ± 1 SD of three technical replicates. Data are representative of three independent experiments from three different healthy donors. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. Results in this figure are from the same experiments as in Figure 12E. Each set of seven bars, from left to right, represents data for EGFRt, CD19, Leu16, rituximab, rituximab.AA, RFR-LCDR, and RFR-LCDR.AA, respectively. [Figure 24]Figures 24A-B. CAR-T cell collection from tumor-carrying mice for transcriptome and epigenetic profiling. NSG mice were intravenously injected with 0.5 × 10⁶ firefly luciferase-expressing Raji cells, and 6 days later, 2.85 × 10⁶ CAR+ T cells were injected. CAR+ T cells were collected from tumor-carrying mice 9 days after T cell injection (n=2 in each group). Only a small number of mock-transduced (EGFRt only) T cells were collected from the mice, which was consistent with the lack of T cell proliferation in the absence of antigen recognition. However, this insufficient cell collection resulted in low read counts in RNA-seq and ATAC-seq, which hindered reliable data analysis. Consequently, these samples were excluded from the analysis shown in Figures 14, 14, and 25. (A) Schematic diagram of the in vivo experiment. (B) Tumor progression monitored by bioluminescence imaging. [Figure 25] Figure 25. RNA-seq reveals significant transcriptome differences among CD20 CAR-T cell variants recovered from tumor-carrying mice. Heatmap of genes (FDR<0.05) differentially expressed in ANOVA comparison of Leu16 CAR-T cells, rituximab CAR-T cells, RFR-LCDR CAR-T cells, and RFR-LCDR.AA CAR-T cells. Each column represents one mouse, and two biological replicates were analyzed for each treatment group. Each row is scaled from a maximum of 1 to a minimum of 0 to highlight the relative expression of each gene. [Figure 26] Figure 26. The T cell subset distribution did not vary significantly among CAR-T cells in ex vivo cultures. Subtypes of CAR-T cells in ex vivo cultures were determined by staining for CD45RA and CD62L in the absence of antigen stimulation. CD45RA+CD62L+, CD45RA-CD62L+, CD45RA-CD62L-, and CD45RA+CD62L- represent naive cell type, central memory cell type, effector memory cell type, and effector cell type, respectively. [Figure 27A]Figures 27A-C. (A) CAR surface expression (top) and transduction marker expression (bottom) detected via antibody staining of IgG4 extracellular spacer (Fc) and EGFRt from one donor. Positive percentage and median fluorescence intensity (MFI) for each sample are noted. (B) CAR surface staining data by %FC+ and MFI for T cells generated from six different healthy donors are shown. (C) CD20 CAR CD8+ T cell enlargement during ex vivo culture with exogenous cytokines IL-2 and IL-15. Fold changes in total viable cell count between day 2 and day 14 are shown. Each data point represents one donor, and data for four donors are shown for each construct. No statistically significant differences were detected between any of the donors by unpaired two-sided two-sample Student's t-test. [Figure 27B] See the explanation in Figure 27A. [Figure 27C] See the explanation in Figure 27A. [Figure 28] Figures 28A-C. (A) Flow cytometry analysis of CD20 expression levels in Raji and K562 cell lines. (B) 24-hour lysis assay of CD20 CAR-TN / M cells against target cells with fluctuating CD20 expression levels at three effector-versus-target (E:T) ratios. Data bars show the mean ± 1 standard deviation (SD) of three technical replicates. No statistically significant differences were observed by unpaired two-sided two-sample Student's t-test. (C) Cytotoxicity and proliferation of CAR CD8+ T cells during repeated antigen challenge by Raji tumor cells. CD20 CAR-T cells were challenged with Raji cells every two days at an E:T ratio of 2:1, and the number of viable Raji and CAR-T cells was quantified by flow cytometry. The data shown are the mean of three technical replicates with error bars showing ± 1 SD. Results from three independent experiments from three different healthy donors are shown. [Figure 29]Figures 29A–D. (A, B) Upregulation of activation and depletion markers and (C, D) T cell proliferation in antigen-independent CAR signaling or rituximab-derived CD20 CARs in the absence of antigen stimulation. Tonic signaling in rituximab-based CAR-T cells was observed in both CD8+ cells (A, C) and naive / memory T (TN / M) cells sorted by CD14- / CD25- / CD62L+ phenotypes. Each set of seven bars in A–D, from left to right, represents data for EGFRt, CD19, GD2, Leu16, rituximab, GA101, and ofatumumab, respectively. [Figure 30] Figure 30. Metabolic flow rates of rituximab-derived CD20 CARs. Each set of four bars, from left to right, represents data for EGFRt, CD19, Leu16, and rituximab, respectively. [Figure 31] Figure 31A-B. (A) CAR-expressing Jurkat cells were stained with an anti-Fc antibody conjugated with DyLight405 in the absence of antigen stimulation and imaged using a confocal microscope. (B) Jurkat cells transduced by a CAR-HaloTag fusion protein were stained with the red fluorescent dye tetramethylrhodamine (TMR) in the absence of antigen stimulation and imaged using a confocal microscope. [Figure 32] Figure 32A-B. (A) NSG mice were intravenously (iv) injected with 0.5 × 10⁶ firefly luciferase-expressing Raji cells, and 6 days later treated with iv delivery of 5 × 10⁶ CD8+ CD20-targeted CAR-T cells. Tumor progression was monitored by bioluminescence imaging (n=6 mice per group). The minimum and maximum values ​​on the radiance scale are 5 × 10⁴ and 1 × 10⁷, respectively. (B) Mean radiance (p / sec / cm² / sr) of individual animals for each test group. The black dotted line indicates day 12 after T cell injection, when the rituximab CAR-T cell group began to rapidly lose tumor control. The endpoints in each trace represent the humane endpoint for each animal. [Figure 33]Figures 33A-C. (A) Schematic diagram of an alanine insertion variant of a rituximab-based CAR. (B) CAR surface expression (top) and transduction marker expression (bottom) detected by antibody staining of IgG4 extracellular spacer (Fc) and EGFRt of the CAR from a single donor, respectively. Positive percentage and median fluorescence intensity (MFI) for each sample are noted. (C) CAR surface staining data by %Fc+ and MFI for T cells generated from four different T cell production runs. [Figure 34] Figures 34A-B. (A) Tumor necrosis factor (TNF)α production by CAR-T cells was measured 7 days after DynaBeads removal in the absence of antigen stimulation. Results from one bulk CD8+ T cell donor and one TN / M cell donor are shown. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (B) Proliferation of CAR+ CD8+ cells and TN / M cells stained with CTV dye was assayed after 4 days of culture in the absence of target cells or exogenous cytokines. Results from one bulk CD8+ T cell donor and one TN / M cell donor are shown. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. [Figure 35] Figures 35A-B. (A) 24-hour lysis assay of rituximab CAR-TN / M cells against target cells with fluctuating CD20 expression levels at different E:T ratios. Data bars show the mean ± 1 SD of three technical replicates. No statistically significant differences were observed by unpaired two-sided two-sample Student's t-test. (B) Cytotoxicity and proliferation of CAR-T cells during repeated antigen challenge. CD20 CAR-T cells were challenged with Raji tumor cells at a 2:1 E:T ratio every two days, and the number of viable Raji cells and CAR-T cells was quantified by flow cytometry. The data shown are the mean of three technical replicates with error bars indicating ± 1 SD. Results from four independent experiments using T cells from four different healthy donors are shown. [Figure 36]Figures 36A-C. (A) Schematic diagram of a GD2 CAR construct in which 0-4 alanine molecules are inserted between the CD28 transmembrane domain and the cytoplasmic domain. (B) NSG mice were intravenously injected with 3.5 × 10⁶ firefly luciferase-expressing CHLA-255 cells, and 17 days later, 2 × 10⁶ GD2 CAR-T cells were intravenously injected. Tumor progression was monitored by bioluminescence imaging. (C) The frequency of human CD45+EGFRt+ cells in the liver and spleen collected from mice at the time of euthanasia was quantified by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. The endpoints for each trace represent the humane endpoints for each animal. [Figure 37A] Figures 37A-B. (A) CAR surface expression (top) and transduction efficiency (bottom) were quantified by antibody staining of Fc and EGFRt. (B) CAR surface staining data by %Fc+ and MFI for T cells generated from 10 different healthy donors are shown. [Figure 37B] See the explanation in Figure 37A. [Figure 38] Figures 38A-B. The RFR-LCDR hybrid CD20 CAR exhibits antigen-independent CAR signaling, resulting in (A) upregulation of activation and depletion markers and (B) T cell proliferation in the absence of antigen stimulation. CD19 CAR and GD2 CAR were included as negative and positive controls for tonic signaling, respectively. Each set of seven bars, from left to right, represents data for EGFRt, CD19, GD2, Leu16, rituximab, RFR-LCDR, and LFR-RCDR, respectively. [Figure 39] Figure 39. The binding dynamics parameters of scFv for Leu16, rituximab, and RFR-LCDR were measured by biolayer interferometry. KD: equilibrium dissociation constant; ka: association constant; kdis: dissociation constant; mean: mean of 3 runs; SD: standard deviation of 3 runs. A two-tailed Student's t-test was used to calculate the shown p-values. [Figure 40]Figures 40A-B. (A) RFR-LCDR.AA is a CAR construct containing RFR-LCDR scFv and two alanine inserts between the CD28 transmembrane domain and the cytoplasmic domain. This CAR exhibits an antigen detection threshold similar to other CD20 CARs in vitro. (B) CARs containing hybrid scFv sequences and / or alanine inserts remain cluster-free on the cell surface. Jurkat cells transduced with CAR-HaloTag fusion proteins were stained with TMR red fluorescent dye and imaged by confocal microscopy. CAR molecules are uniformly distributed on the cell surface in the absence of antigen stimulation. [Figure 41] Figures 41A-C. (A) Expression of activation and depletion markers by CAR-TN / M cells in the absence of CD20 antigen stimulation was evaluated 11 days after DynaBeads removal. Data bars show the mean ± 1 SD of three technical replicates. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (B) Proliferation of CAR+ TN / M cells stained with CTV dye was assayed after 4 days of culture in the absence of target cells or exogenous cytokines. Data shown in the histogram correspond to donor 1 in the bar graph. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. (C) Production of IFNγ, TNFα, and IL-2 by CAR+ TN / M cells in the absence of antigen stimulation. Cytokine concentrations in the supernatant of cells cultured for 48 hours in the absence of exogenous cytokines were measured by ELISA. The data bars show the mean ± 1 SD of the technical 3 replicates. *p<0.05, **p<0.01, ***p<0.001, ns not statistically significant. Each set of seven bars, from left to right, represents data for EGFRt, CD19, Leu16, rituximab, rituximab.AA, RFR-LCDR, and RFR-LCDR.AA, respectively. The data in (B), from top to bottom, represent EGFRt, CD19, Leu16, rituximab, rituximab.AA, RFR-LCDR, and RFR-LCDR.AA. [Figure 42]Figure 42. CAR tonic signaling. Each set of seven bars, from left to right, represents data for EGFRt, CD19, Leu16, rituximab, rituximab.AA, RFR-LCDR, and RFR-LCDR.AA, respectively. [Figure 43-1] Figures 43A-H. Tonic signaling CARs exhibit different transcriptional profiles, showing (A) increased mitochondrial protein translation, (B) increased antigen presentation, (C) increased MYC signaling, (D) increased MTORC signaling, (E) increased TNFα signaling, (F) diverse interferon responses, (G) enrichment of gene signatures associated with effector T cell subtypes (in contrast to memory T cell subtypes), and (H) increased cell cycle activity. For heatmaps containing more genes than the available space, the names of the genes shown in the heatmap are listed sequentially next to each map. [Figure 43-2] See the explanation in Figure 43-1. [Figure 43-3] See the explanation in Figure 43-1. [Figure 43-4] See the explanation in Figure 43-1. [Figure 43-5] See the explanation in Figure 43-1. [Figure 44-1]Figures 44A-D. RFR-LCDR hybrid CD20 CARs exhibit superior tumor clearance in vivo. (A) Schematic diagram of the animal study. 500,000 firefly luciferase-expressing Raji lymphoma cells were engrafted into NSG mice and treated with two T-cell administrations. The animals were re-challenged with a second administration of tumor cells 55 days after the first T-cell treatment. (B) Tumor progression was monitored through bioluminescence imaging. (C) Survival curves are shown as Kaplan-Meier curves. The results show that the hybrid CAR is superior to both the parental CD20 CAR and CD19 CAR in tumor clearance and long-term recurrence prevention. (D) The frequency of CAR-expressing human T cells in peripheral blood 23 days after the first T-cell injection was measured by flow cytometry. Addition of two alanine residues to the CAR improved the rituximab-based receptor, thus confirming the previous data shown in Figure 5. Combining hybrid CARs with two alanine insertions further accelerated the rate of early tumor clearance and resulted in a significant increase in T cell persistence compared to hybrid CARs without alanine insertions. This suggests that combining scFv modification and alanine insertion strategies can lead to synergistic improvements in CAR function. [Figure 44-2] See the explanation in Figure 44-1. [Figure 45A]Figures 45A-C. (A) Volcano plots of differentially expressed genes in comparison between rituximab CAR-T cells and Leu16 CAR-T cells (left), RFR-LCDR CAR-T cells (center), or RFR-LCDR.AA CAR-T cells (right) based on RNA sequencing. All differentially expressed genes are plotted in gray. Genes with FDR < 0.05 and at least 2x upregulation or downregulation (log2FC > 1 or log2FC < -1) are shown as red dots. Genes that appear in all three sets of pairwise comparisons with log2FC > 1 and FDR < 0.05 or log2FC < -1 and FDR < 0.05 are marked. (B) Genomic browser files of differentially accessible regions based on ATACseq at the ATP9A, KIR2DL3, KIR2DL1, and KIR3DL1 loci of Leu16 CAR-T cells, rituximab CAR-T cells, RFR-LCDR CAR-T cells, and RFR-LCDR.AA CAR-T cells. (C) Gene set enrichment analysis (GSEA) was performed on the RNA-seq data obtained as shown in Figure 5A. A summary of the results in pathways related to T cell phenotype and function is shown in BubbleGUM map format. [Figure 45B] See the explanation in Figure 45A. [Figure 45C] See the explanation in Figure 45A. [Modes for carrying out the invention]

[0067] Exemplary Description Chimeric antigen receptors contain a single-chain variable fragment (scFv) having a heavy chain (VH) and a light chain (VL), each containing a framework region (FR) adjacent to a complementarity-determining region (CDR). The framework region is considered a major determinant of the scFv structure. Certain FR sequences, when incorporated as part of the scFv portion of a CAR, may induce tonic signaling, which has been proposed to be likely due to their effect on inducing CAR cluster formation. Previous studies have shown that the FR sequence of a tonic signaling CAR (GD2) can be combined with the CDR sequence of a non-tonic signaling CD19 CAR, resulting in a hybrid CAR that exhibits tonic signaling. From these results, it was hypothesized that incorporating the FR sequence of a tonic signaling CAR is generally undesirable in the development of new CAR molecules (Long et al. Nature Medicine, 2015. 21(6):581-590). Surprisingly, the inventors have found that incorporating FRs derived from a tonic signaling CAR results in a CAR with superior properties. Accordingly, the compositions and methods of this disclosure provide a redesigned CAR, e.g., CD20 CAR, having increased in vivo efficacy.

[0068] I. Definition The peptides of this disclosure relate to peptides containing chimeric antigen receptors, or CARs. CARs are engineered receptors that can be implanted with arbitrary specificity onto immune effector cells. In some cases, these receptors are used to implant the specificity of monoclonal antibodies onto T cells. Receptors are called chimeric because they are composed of parts derived from various sources.

[0069] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to gene products.

[0070] "Homologie" or "identity" refers to sequence similarity between two peptides or two nucleic acid molecules. Identity can be determined by comparing the positions in each sequence that can be aligned for comparison. If the positions in the compared sequences are occupied by the same base or amino acid, the molecules share sequence identity at that position. The degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 60%, less than 50%, less than 40%, less than 30%, or less than 25% identity with one of the sequences of this disclosure.

[0071] In this specification, terms such as “amino moiety,” “N-terminus,” and “amino terminus” are used to refer to the order of regions in a polypeptide. Furthermore, when something is at the N-terminus of a region, it is not necessarily at the end (or edge) of the entire polypeptide, but only at the N-terminus of the region or domain. Similarly, in this specification, terms such as “carboxy moiety,” “C-terminus,” and “carboxy terminus” are used to refer to the order of regions in a polypeptide, and when something is at the C-terminus of a region, it is not necessarily at the end (or edge) of the entire polypeptide, but only at the C-terminus of the region or domain.

[0072] The terms “polynucleotide,” “nucleic acid,” and “oligonucleotide” are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modifications to the nucleotide structure, where present, can be given before or after the assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeling components. This term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or requested, any aspect of the present invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0073] A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” that “codes” a particular protein is a nucleic acid molecule that, when under the control of an appropriate regulatory sequence, is transcribed in vitro or in vivo and optionally also translated into a gene product, such as a polypeptide. The coding region may exist in cDNA, genomic DNA, or RNA form. When existing in DNA form, the nucleic acid molecule may be single-stranded (i.e., sense strand) or double-stranded. The boundaries of the coding region are determined by a start codon at the 5' (amino) end and a translation termination codon at the 3' (carboxy) end. Genes may include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically located 3' relative to the gene sequence.

[0074] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies, and antibody fragments, which may be derived from humans, mice, humanized organisms, chimeric organisms, or other species. A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies directed against a specific antigenic site.

[0075] "Antibody or functional fragment thereof" refers to 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, peptidebodies, 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 antigen-binding regions of antibodies, such as Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. The term scFv refers to a single-stranded Fv antibody in which the variable domains of the heavy and light chains of a traditional double-stranded antibody are linked to form a single chain.

[0076] As used herein, the term “binding affinity” refers to the equilibrium constant of the reversible binding of two agents, and is expressed as the 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 within that range). As used herein, the term “binding strength” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactivity” and “preferential binding” are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0077] The term "bonding" refers to the direct association between two molecules, including, for example, interactions such as salt bridges and water bridges, through covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions.

[0078] The terms "individual," "subject," and "patient" are used interchangeably and can refer to either a human or a non-human.

[0079] The terms “lower,” “reduced,” “decrease,” “decrease,” or “inhibit” are all used herein to generally mean a statistically significant reduction. However, to avoid any doubt, “lower,” “reduced,” “decrease,” “decrease,” or “inhibit” means a reduction of at least 10% compared to a reference level, for example, a reduction of 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% compared to a reference level, or a reduction up to 100% and including 100% (i.e., no level compared to a reference sample), or any reduction between 10% and 100%.

[0080] The terms “increased,” “boosted,” “enhanced,” or “activated” are all used herein to generally mean an increase of a statically significant amount; to avoid any doubt, the terms “increased,” “boosted,” “enhanced,” or “activated” mean an increase of at least 10% compared to a reference level, for example, an increase of 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 an increase up to and including 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level.

[0081] II. Polypeptides A. Signal peptide The polypeptides of this disclosure may include signal peptides. “Signal peptide” refers to a peptide sequence that directs the transport and localization of an intracellular protein to, for example, a specific organelle (such as the endoplasmic reticulum) and / or the cell surface. In some embodiments, the signal peptide directs the nascent protein into the endoplasmic reticulum, which is essential if the receptor is to be glycosylated and immobilized on the cell membrane. Generally, the signal peptide naturally bound to the most amino-terminal component is used (for example, in scFv having a light-linker-heavy-chain orientation, the natural signal on the light chain is used).

[0082] In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. In some embodiments, the limiting site is located at the carboxyl terminus of the signal peptide to facilitate cleavage.

[0083] B. Antigen-binding domain The polypeptides of this disclosure may comprise one or more antigen-binding domains. “Antigen-binding domain” describes a region of the polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, the antigen-binding domain is a single-stranded variable fragment (scFv) based on one or more antibodies (e.g., CD20 antibodies). In some embodiments, the antigen-binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, where the VH and VL regions lie on the same polypeptide. In some embodiments, the antigen-binding domain comprises a linker between the VH and VL regions. The linker may enable the antigen-binding domain to form a desirable structure for antigen binding.

[0084] The variable regions of the antigen-binding domains of the polypeptides of this disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more antibody binding properties (e.g., affinity). The term "CDR" refers to complementarity-determining regions based on a portion of the variable chain in immunoglobulins (antibodies) and T cell receptors, respectively, produced by B cells and T cells, which bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T cell receptors are found in CDRs, these regions are sometimes called hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties of interest can be evaluated by appropriate in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically involving changes to one, two, three, four, or five or fewer residues within the CDR region. Mutations may be amino acid substitutions, additions, or deletions.

[0085] Framework modification can be performed on antibodies to reduce immunogenicity, for example, by "reverse mutagenes" one or more framework residues into the corresponding germline sequences.

[0086] The antigen-binding domain may also be multispecific or polyvalent by polymerizing the antigen-binding domain with a pair of VH and VL regions that bind to either the same antigen (polyvalent) or different antigens (multispecific).

[0087] The binding affinity of the antigen-binding region, such as the variable region (heavy chain and / or light chain variable region), or the CDR, is at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13 M may also be used. In some embodiments, the antigen-binding region, such as the variable region (heavy chain and / or light chain variable region), or the K of the CDR. D at least 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or 10 -13 It may also be M (or any range that can be derived within it).

[0088] Binding affinity, K A , or K D This can be determined by methods known in the art, for example, by surface plasmon resonance (SRP) based biosensors, by the binding equilibrium exclusion method (KinExA), by optical scanners for microarray detection based on the difference in oblique incidence reflectance (OI-RD) of polarization modulation, or by ELISA.

[0089] In some embodiments, polypeptides containing a humanized binding domain have better binding affinity and / or expression levels in host cells than polypeptides containing a non-humanized binding domain, such as mouse-derived binding domains, at least equivalent to or better than 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% of those containing a humanized binding domain.

[0090] In some embodiments, the framework regions of the human framework, such as FR1, FR2, FR3, and / or FR4, are at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 1 02, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 13 3, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 ,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195, 196, 197, 198, 199, or 200, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 ,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 ,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,12 6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,It may have 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range within that range) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions, individually or collectively.

[0091] In some embodiments, the framework regions of the mouse framework, such as FR1, FR2, FR3, and / or FR4, are at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 1 02, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 13 3, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 ,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195, 196, 197, 198, 199, or 200, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 ,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 ,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,12 6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,It may have 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 (or any derivable range within that range) amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions, individually or collectively.

[0092] The substitution is in the heavy chain or light chain variable region FR1, FR2, FR3, or FR4, specifically in the 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100th place.

[0093] C. Extracellular spacer The extracellular spacer may ligate the antigen-binding domain to the transmembrane domain. In some embodiments, the hinge is sufficiently mobile to allow the antigen-binding domain to orient in various directions to facilitate antigen binding. In one embodiment, the spacer includes an IgG-derived hinge region. In some embodiments, the spacer includes, or further includes, the CH2CH3 region and a portion of CD3 of immunoglobulin. In some embodiments, the CH2CH3 region may be a modification of L235E / N297Q or L235D / N297Q, or a CH2CH3 region with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity. In some embodiments, the spacer is IgG4-derived. The extracellular spacer may include a hinge region.

[0094] As used herein, the term “hinge” refers to a mobile polypeptide connector region (also referred to herein as a “hinge region”) that provides structural mobility and spacing to adjacent polypeptide regions, and may consist of a natural or synthetic polypeptide. A “hinge” derived from an immunoglobulin (e.g., IgG1) is generally defined as extending from Glu216 to Pro230 in human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond in the same position. Hinge regions may be naturally occurring or non-natural, including but not limited to modified hinge regions as described herein, such as those described in U.S. Patent No. 5,677,425, incorporated herein by reference. Hinge regions may include complete hinge regions derived from antibodies of a different class or subclass than those of the CH1 domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide similar functions in terms of providing mobility and spacing to adjacent regions.

[0095] Extracellular spacers are present in at least 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 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, 290, 300, 325, 350, or 400 amino acids, at most 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 2 11, 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, 290, 300, 325, 350, or 400 amino acids, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 1 00, 110, 119, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 22 0, 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, 290, 300,The extracellular spacer can have a length of 325, 350, or 400 amino acids (or any derivable range thereof). In some embodiments, the extracellular spacer consists of or includes a hinge region derived from immunoglobulin (e.g., IgG). The amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162 and Huck et al. (1986) Nucl. Acids Res.

[0096] The length of the extracellular spacer may affect the signaling activity of CARs and / or the growth characteristics of CAR-T cells in response to antigen-stimulated CAR signaling. In some embodiments, shorter spacers are used, such as 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or less than 10 amino acids. In some embodiments, 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, 22 Longer spacers, such as those with 9, 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 increasing growth in vivo or in vitro.

[0097] As a non-limiting example, an immunoglobulin hinge region may contain one of the following amino acid sequences:

[0098] Table: Exemplary Hinge Area TIFF0007834345000001.tif85160

[0099] The extracellular spacer may contain the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4. The extracellular spacer may also contain one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, the hinge region may contain the sequence The His229 of the human IgG1 hinge can be replaced with Tyr, as shown in TIFF0007834345000002.tif4128.

[0100] The extracellular spacer may contain an amino acid sequence derived from human CD8; for example, the hinge region may contain an amino acid sequence: This may include TIFF0007834345000003.tif4151 or its variants.

[0101] The extracellular spacer may include, or may further include, a CH2 region. An example CH2 region is: The filename is TIFF0007834345000004.tif19160. The extracellular spacer may include, or may further include, a CH3 region. An example CH3 region is: The filename is TIFF0007834345000005.tif19160.

[0102] If the extracellular spacer contains multiple parts, 0 to 50 amino acids may be present anywhere between the various parts. For example, between the hinge and the CH2 or CH3 region, or between the CH2 and CH3 regions if both are present, at least 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, and at most 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 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 within that range) may be 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, excluding all other domains or regions, but there may be further amino acids that are not part of the identifiable region.

[0103] D. Transmembrane domain The polypeptides of this disclosure may include transmembrane domains. In some embodiments, the transmembrane domain is a hydrophobic α-helix spanning the membrane. Different transmembrane domains may result in different receptor stabilities.

[0104] In some embodiments, the transmembrane domain is inserted between an extracellular spacer and a cytoplasmic region. In some embodiments, the transmembrane domain is inserted between an extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is located between the transmembrane domain and one or more costimulatory regions.

[0105] Any transmembrane domain that provides a polypeptide insertion 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-ζ, CD134, or CD7.

[0106] Examples of transmembrane domains useful in any aspect of this disclosure include those listed in the following table.

[0107] Table: Exemplary transmembrane domain sequences TIFF0007834345000006.tif68160

[0108] E. Cytoplasmic region The receptors of this disclosure cluster after antigen recognition, and signals can be transmitted to cells through the cytoplasmic region. In some embodiments, the costimulatory domains described herein are part of the cytoplasmic region. In some embodiments, the cytoplasmic region includes an intracellular signaling domain. The intracellular signaling domain may include a primary signaling domain and one or more costimulatory domains.

[0109] The cytoplasmic and / or co-stimulatory regions suitable for use in the polypeptides of this disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by cells; altered transcription of a target gene; altered protein activity; altered cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of cellular signaling responses, etc.) in response to activation via the binding of an 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 as described herein. In some embodiments, the cytoplasmic region includes a DAP10 / CD28 type signaling chain.

[0110] Cytoplasmic regions suitable for use in polypeptides of this disclosure include intracellular signaling polypeptides containing an immune receptor-activated tyrosine motif (ITAM). The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acids. In some cases, the cytoplasmic region contains 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, and the first and second examples of the ITAM motif are separated from each other by 6 to 8 amino acids, e.g., (YX1X2(L / I))(X3)n(YX1X2(L / I)), where n is an integer from 6 to 8, and each of the 6 to 8 X3 can be any amino acid.

[0111] A suitable cytoplasmic region may be an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Therefore, a suitable end domain does not need to contain the entire sequence of the protein from which it originates. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, DAP10, FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3-ζ; and CD79A (antigen receptor complex-associated protein α chain).

[0112] Exemplary cytoplasmic regions are known in the art. The cytoplasmic regions shown below also provide examples of regions that can be incorporated into the CAR of this disclosure.

[0113] In some embodiments, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. In some embodiments, the cytoplasmic region is derived from FCER1G (FCRG; Fcε receptor Iγ chain; Fc receptor γ- chain; fc-εR1-γ; fcRγ; fceRIγ; also known as high-affinity immunoglobulin ε receptor subunit γ; immunoglobulin E receptor, high-affinity, γ chain, etc.). In some embodiments, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length FCER1G amino acid sequence.

[0114] In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3δ 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, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length CD3δ amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3ε chain (also known as CD3e, CD3ε; 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, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length CD3ε amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3γ chain (also known as CD3G, CD3γ, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some embodiments, a suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length CD3γ amino acid sequence. In some embodiments, the cytoplasmic region is derived from the T cell surface glycoprotein CD3ζ chain (also known as CD3Z, CD3ζ, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).

[0115] In some embodiments, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length CD3ζ amino acid sequence. In some embodiments, the cytoplasmic region is derived from CD79A (also known as the B cell antigen receptor complex-associated protein α chain; CD79a antigen (immunoglobulin-associated α); MB-1 membrane glycoprotein; ig-α; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.). In some embodiments, the suitable cytoplasmic region may include an ITAM motif-containing portion of the full-length CD79A amino acid sequence.

[0116] Specific exemplary cytoplasmic regions are known in the art and are further shown in the table below.

[0117] Table: Cytoplasmic region TIFF0007834345000007.tif60166TIFF0007834345000008.tif234166TIFF0007834345000009.tif145166

[0118] F. Co-stimulation area Non-limited examples of suitable co-stimulatory regions, including those located in the cytoplasm, include, but are not limited to, polypeptides derived from 4-1BB(CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0119] The co-stimulatory region may have a length of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, at most 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any derivable range within that range. In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA, etc.). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, and TXGP1L). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0120] Specific exemplary costimulatory domains are shown by the following amino acid sequence.

[0121] Table: Co-stimulatory domains TIFF0007834345000010.tif123166

[0122] G. Detected peptide In some embodiments, the polypeptides described herein may further comprise detection peptides. Suitable detection peptides include hemagglutinins (HA; e.g., YPYDVPDYA (SEQ ID NO: 96)); FLAG (e.g., DYKDDDDK (SEQ ID NO: 97)); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 98)), etc. Other suitable detection peptides are known in the art.

[0123] H. Peptide Linker In some embodiments, the polypeptides of this disclosure include peptide linkers (sometimes referred to as linkers). Peptide linkers may be used to separate any of the peptide domains / regions described herein. For example, linkers may be between a signal peptide and an antigen-binding domain, between the VH and VL of an antigen-binding domain, between an antigen-binding domain and a peptide spacer, between a peptide spacer and a transmembrane domain, adjacent to a costimulatory region, or on the N or C region of a costimulatory region, and / or between a transmembrane domain and an endodomain. Peptide linkers may have any of a variety of amino acid sequences. Domains and regions can be linked by peptide linkers, which are generally mobile, although other chemical bonds are not excluded. Linkers may be peptides about 6 to about 40 amino acids long, or about 6 to about 25 amino acids long. These linkers can be prepared by using oligonucleotides encoding synthetic linkers to couple proteins.

[0124] A peptide linker with some degree of mobility can be used. Keeping in mind that a suitable peptide linker generally has a sequence that results in a mobile peptide, the peptide linker may have virtually any amino acid sequence. The use of small amino acids such as glycine and alanine is useful for creating mobile peptides. The creation of such sequences is commonplace for those skilled in the art.

[0125] Suitable linkers can be easily selected and can be of any suitable length, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, or 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0126] Suitable linkers can be easily selected and are of various lengths suitable, for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, 1 to 20 amino acids (e.g., Gly), 2 to 15 amino acids, 3 to 12 amino acids, or 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0127] Examples of mobile linkers include glycine-serine polymers, such as (G)n, e.g., (GS)n, (GSGGS)n (SEQ ID NO: 99), (G4S)n, and (GGGS)n (SEQ ID NO: 100), where n is an integer of at least 1. In some embodiments, n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range within that range). Glycine-alanine polymers, alanine-serine polymers, and other mobile linkers are known in the art. Glycine polymers and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore act as neutral tethers between components. Glycine polymers can be used; glycine accesses a significantly larger phi-psi space than alanine and is far less restrictive than residues with longer side chains. Exemplary spacers may include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO: 101), GGSGG (SEQ ID NO: 102), GSGSG (SEQ ID NO: 103), GSGGG (SEQ ID NO: 104), GGGSG (SEQ ID NO: 105), GSSSG (SEQ ID NO: 106), etc.

[0128] In a further embodiment, the linker includes (EAAAK)n (SEQ ID NO: 107), where n is an integer of at least 1. In some embodiments, n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range thereof).

[0129] I. Additional modifications and polypeptide enhancements Additionally, the polypeptides of this disclosure may be chemically modified. The glycosylation of the polypeptide can be altered, for example, by modifying one or more glycosylation sites within the polypeptide sequence to increase the polypeptide's affinity for an antigen (U.S. Patents 5,714,350 and 6,350,861).

[0130] Regions or fragments of the polypeptides of this disclosure are 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 ,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124 ,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,1 It is intended that the amino acid sequence may have, at least, or at most, 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 or more amino acid substitutions, consecutive amino acid additions, or consecutive amino acid deletions. Alternatively, a region or fragment of the polypeptide of this disclosure corresponds to any of SEQ ID NO: 1 to 144, specifically 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or any derivable range within that range) are identical, at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or any derivable range within that range) It may have an amino acid sequence that is identical to, or at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% (or any derivable range within that) the SEQ ID NO: 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 ,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,11 7, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450 , 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500th place (1st place is SEQ ID) Starting from the N-terminus of NO, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 ,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,13 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、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、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464 , containing an amino acid region of 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more consecutive amino acids. The polypeptides of this disclosure may contain 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acids, or SEQ ID NO:1~144, at least 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 1 68, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 19 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 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, 300, 400, 500, 550, 600 consecutive amino acids, or more At most, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 1, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 1 93, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 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, 300, 400, 500, 550, 600 or more consecutive amino acids, or any derivable range within t...

Claims

1. A polypeptide comprising an anti-CD20 single-chain variable fragment (scFv), The light chain variable region (VL) includes, in order from the amino proximal terminus to the carboxyl proximal terminus, light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). Includes, Here, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs. 29, 30, 31, 22, 12, 13, and 14, respectively; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of SEQ ID NOs. 26, 27, 28, 18, 9, 10, and 11, respectively. The aforementioned polypeptide.

2. The polypeptide according to claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

6.

3. The polypeptide according to claim 1, wherein VH is amino-proximal to VL, or VH is carboxyl-proximal to VL.

4. The polypeptide according to claim 1, comprising the anti-CD20 single-chain variable fragment (scFv) and a cytoplasmic region including a transmembrane domain and a primary intracellular signaling domain.

5. The polypeptide according to claim 4, wherein the transmembrane domain is derived from the α or β chain of a T cell receptor, CD28, CD3ε (epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154.

6. The polypeptide according to claim 4, wherein the cytoplasmic region further comprises one or more costimulatory domains.

7. The polypeptide according to claim 6, wherein the one or more co-stimulatory domains include co-stimulatory domains derived from one or more of 4-1BB (CD137), CD28, IL-15Rα, OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), or ICOS (CD278).

8. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 33 or 42.

9. The polypeptide according to claim 4, further comprising a twisted linker between the transmembrane domain and the cytoplasmic region.

10. The polypeptide according to claim 9, wherein the twisted linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alanine residues.

11. A polypeptide comprising a CAR containing, in order from the amino proximal terminus to the carboxyl proximal terminus, an anti-CD20 scFv, a CD28 transmembrane domain, a twisted linker, and a cytoplasmic region containing a CD28 costimulatory domain and a CD3ζ intracellular signaling domain, wherein the twisted linker contains 1 to 12 alanine residues, The CD20 scFv is, The light chain variable region (VL) includes, in order from the amino proximal terminus to the carboxyl proximal terminus, light chain framework region 1 (LFR1), light chain complementarity determination region 1 (LCDR1), light chain framework region 2 (LFR2), light chain complementarity determination region 2 (LCDR2), light chain framework region 3 (LFR3), light chain complementarity determination region 3 (LCDR3), and light chain framework region 4 (LFR4); and The heavy chain variable region (VH) includes, in order from the amino proximal end to the carboxyl proximal end, heavy chain framework region 1 (HFR1), heavy chain complementarity determination region 1 (HCDR1), heavy chain framework region 2 (HFR2), heavy chain complementarity determination region 2 (HCDR2), heavy chain framework region 3 (HFR3), heavy chain complementarity determination region 3 (HCDR3), and heavy chain framework region 4 (HFR4). Including; Here, LFR1, LFR2, LFR3, LFR4, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs. 29, 30, 31, 22, 12, 13, and 14, respectively; and HFR1, HFR2, HFR3, HFR4, HCDR1, HCDR2, and HCDR3 each contain the amino acid sequences of SEQ ID NOs. 26, 27, 28, 18, 9, 10, and 11, respectively. The aforementioned polypeptide.

12. A nucleic acid comprising a sequence encoding the polypeptide described in any one of claims 1 to 11.

13. A cell or cell population comprising the nucleic acid described in claim 12.

14. The cells or cell population according to claim 13, which are T cells, natural killer (NK) cells, naive memory T cells, natural killer T cells (NKT), invariant natural killer T cells (iNKT), stem cells, lymphocyte progenitor cells, peripheral blood mononuclear cells (PBMCs), bone marrow cells, fetal hepatocytes, embryonic stem cells, hematopoietic stem cells or hematopoietic progenitor cells (HSPCs), umbilical cord blood cells, or induced pluripotent stem cells.

15. A cell population according to claim 13 for use in the treatment of cancer.

16. The cell population according to claim 15, wherein the cancer includes lymphoma or neuroblastoma.

17. The cell population according to claim 15, wherein the cancer includes brain cancer.

Images