Proteinase 3 (PR3) Chimeric Autoantibody Receptor T Cells and Related Methods and Uses

Abstract

Provided herein are cell and protein therapeutics comprising proteinase 3 (PR3) antibody binding domain, for example, wild-type PR3 protein or mutant PR3 protein, and methods of using the same.Also provided herein are chimeric autoantibody receptors (CAARs) comprising extracellular PR3 antibody binding domains.Among the CAARs provided are CAARs whose extracellular PR3 antibody binding domains are wild-type or mutant PR3 proteins or fragments thereof that can receive specific binding of anti-PR3 antineutrophil cytoplasmic antibodies (ANCA).Also provided herein are polynucleotides encoding the provided CAARs, genetically engineered cells such as T cells that contain the provided CAARs, and related methods and uses thereof in adoptive cell therapy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 312,809, filed February 22, 2022, entitled "Proteinase 3 (PR3) Chimeric Autoantibody Receptor T Cells and Related Methods and Uses," the contents of which are incorporated by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided in a file named 735042022040SeqList.xml, created on February 18, 2023, and is 304,200 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] The present disclosure provides cell or protein therapeutics comprising proteinase 3 (PR3) antibody binding domains, such as wild-type PR3 or mutant PR3 proteins. The present disclosure also provides chimeric autoantibody receptors (CAARs) comprising extracellular PR3 antibody binding domains. Some of the CAARs provided in the present disclosure are wild-type or mutant PR3 proteins or fragments thereof, in which the extracellular PR3 antibody binding domains can be specifically bound by anti-PR3 antineutrophil cytoplasmic antibodies (ANCA). The present disclosure also provides polynucleotides encoding the provided CAARs, engineered cells, such as T cells, containing the provided CAARs, and related methods and uses thereof in adoptive cell therapy. [Background technology]

[0004] Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a rare autoimmune disease characterized by inflammation and damage to small blood vessels. Neutrophil serine protease proteinase 3 (PR3), found in primary granules of neutrophils, is the major autoantigen of AAV. Autoantibodies against PR3 (ANCA), produced by autoreactive B cells, are crucial to the pathogenesis of AAV by activating primed neutrophils that damage blood vessels. Improved methods for targeting and killing disease-causing B cells are needed. Embodiments that meet such needs are provided. Summary of the Invention [Means for solving the problem]

[0005] Provided herein is a chimeric autoantibody receptor (CAAR) comprising: (a) an extracellular proteinase 3 (PR3) antibody binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the CAAR further comprises a spacer between the extracellular PR3 antibody binding domain and the transmembrane domain. In some of any of the embodiments, the PR3 antibody binding domain is derived from or a mutant of human PR3 protein. In some of any of the embodiments, the PR3 antibody binding domain is a mature PR3 protein. In some of any of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids shown in SEQ ID NO:1, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO:1. In some of any of the embodiments, the PR3 antibody binding domain is wild-type human PR3. In some of any of the embodiments, the PR3 antibody binding domain is shown in SEQ ID NO:1.

[0006] In some of any of the embodiments, the PR3 antibody binding domain is a mutant PR3 protein comprising an amino acid substitution at a position selected from the group consisting of G4, H44, D91, D175 and S176, relative to the position shown in SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from the group consisting of G4P, H44A, D91N, D175N, S176A and S176C, or a conservative substitution thereof. In some embodiments, the amino acid substitution is selected from the group consisting of G4P, H44A, D91N, D175N, S176A and S176C. In some embodiments, the amino acid substitution is a conservative amino acid substitution of an amino acid substitution selected from the group consisting of G4P, H44A, D91N, D175N, S176A and S176C.

[0007] In some of any of the embodiments, the PR3 antibody binding domain is a mutant PR3 protein that has reduced enzymatic activity compared to a wild-type PR3 protein (eg, the PR3 protein set forth in SEQ ID NO:1).

[0008] Among the embodiments provided are chimeric autoantibody receptors (CAARs) comprising: (a) an extracellular proteinase 3 (PR3) antibody binding domain, where the PR3 antibody binding domain is a mutant PR3 protein having reduced enzymatic activity compared to a wild-type PR3 protein having the sequence set forth in SEQ ID NO:1; (b) a transmembrane region; and (c) an intracellular signaling region.

[0009] In some embodiments, the mutant PR3 protein comprises an amino acid substitution in or near the catalytic triad. In some embodiments, the amino acid substitution is at an active site residue selected from the group consisting of H44, D91 and S176, based on the numbering shown in SEQ ID NO: 1. In some embodiments, the amino acid substitution is selected from the group consisting of H44A, D91N, S176A and S176C. In some of any of the embodiments, the mutant PR3 protein comprises an amino acid substitution at position H44, based on the numbering of the positions in SEQ ID NO: 1.

[0010] In some embodiments, the mutant PR3 protein comprises an amino acid substitution at a position that interferes with the formation of the substrate binding pocket. In some embodiments, the position is G4. In some of the embodiments, the mutant PR3 protein comprises an amino acid substitution at position G4, relative to the numbering of the positions in SEQ ID NO:1. In some of the embodiments, the mutant PR3 protein comprises one, two, three, four or five amino acid substitutions compared to the wild-type PR3 shown in SEQ ID NO:1. In some of the embodiments, the mutant PR3 protein comprises a single amino acid substitution compared to SEQ ID NO:1. In some of the embodiments, the mutant PR3 protein comprises the amino acid substitution G4P.

[0011] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the PR3 antibody binding domain is a mutant PR3 protein comprising the amino acid substitution G4P. In some of the embodiments, the PR3 antibody binding domain comprises the amino acid sequence shown in SEQ ID NO:9. In some of the embodiments, the PR3 antibody binding domain comprises the amino acid sequence showing at least 90% sequence identity with SEQ ID NO:9. In some of the embodiments, the PR3 antibody binding domain is shown in SEQ ID NO:9. In some of the embodiments, the mutant PR3 protein comprises the amino acid substitution H44A.

[0012] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain; (b) a transmembrane region; and (c) an intracellular signaling region, wherein the PR3 antibody binding domain is a mutant PR3 protein comprising the amino acid substitution H44A. In some of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO: 10. In some of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 10. In some of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO: 10. In some of the embodiments, the amino acid sequence of the PR3 antibody binding domain consists of the sequence set forth in SEQ ID NO: 10. In some of the embodiments, the mutant PR3 protein comprises the amino acid substitution D91N. In some of the embodiments, the antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO: 4, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 4. In some of the embodiments, the PR3 antibody binding domain is set forth in SEQ ID NO: 4.

[0013] In some of any of the embodiments, the mutated PR3 protein comprises the amino acid substitution D175N. In some of any of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO:7, or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:7. In some of the embodiments, the PR3 antibody binding domain is set forth in SEQ ID NO:7. In some of the embodiments, the mutated PR3 protein comprises the amino acid substitution S176A or S176C. In some of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO:2, or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:2. In some of the embodiments, the PR3 antibody binding domain is set forth in SEQ ID NO:2. In some of the embodiments, the PR3 antibody binding domain comprises a sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:8. In some of the embodiments, the PR3 antibody binding domain is set forth in SEQ ID NO:8.

[0014] In some of the embodiments, the transmembrane region is or comprises a transmembrane domain from CD4, CD28, or CD8. In some of the embodiments, the transmembrane region is or comprises a transmembrane domain from CD28. In some of the embodiments, the transmembrane region comprises human CD28. In some of the embodiments, the transmembrane region is or comprises SEQ ID NO:26, or is or comprises an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:26. In some of the embodiments, the transmembrane region is set forth in SEQ ID NO:26. In some of the embodiments, the transmembrane region is or comprises a transmembrane domain from CD8a. In some of the embodiments, the transmembrane domain is human CD8a. In some of any of the embodiments, the transmembrane region is or comprises SEQ ID NO:98 or is or comprises an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:98. In some of any of the embodiments, the transmembrane region is set forth in SEQ ID NO:98.

[0015] In some of the embodiments, the intracellular signaling region comprises an intracellular signaling domain capable of inducing a primary activation signal in T cells. In some of the embodiments, the intracellular signaling domain is a domain derived from a T cell receptor (TCR) component and / or comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some of the embodiments, the intracellular signaling domain is a cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain. In some of the embodiments, the intracellular signaling domain is a human CD3ζ chain. In some of the embodiments, the intracellular signaling domain comprises a sequence as set forth in SEQ ID NO:28 or an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:28. In some of the embodiments, the intracellular signaling domain is set forth in SEQ ID NO:28.

[0016] In some of the embodiments, the intracellular signaling region further comprises a costimulatory signaling region. In some of the embodiments, the costimulatory signaling region is between the transmembrane region and the intracellular signaling domain. In some of the embodiments, the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some of the embodiments, the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS. In some of the embodiments, the costimulatory signaling region comprises an intracellular signaling domain of 4-1BB. In some of the embodiments, the costimulatory signaling region comprises human 4-1BB. In some of the embodiments, the costimulatory signaling region comprises a sequence as set forth in SEQ ID NO:27, or an amino acid sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27. In some of the embodiments, the costimulatory signaling region is set forth in SEQ ID NO:27.

[0017] In some of the embodiments, the spacer comprises at least a portion of an immunoglobulin or variant thereof. In some of the embodiments, the spacer comprises a hinge region of an immunoglobulin or variant thereof. In some of the embodiments, the hinge region of the immunoglobulin is an IgG4 hinge region. In some of the embodiments, the hinge region is a human IgG4 hinge region, or variant thereof. In some of the embodiments, the spacer comprises a mutant IgG4 hinge region comprising a substitution of amino acids CPSC to CPPC compared to a wild-type IgG4 hinge region. In some of the embodiments, the spacer is less than 15 amino acids or about 15 amino acids in length. In some of the embodiments, the spacer is 12 to 15 amino acids in length. In some of the embodiments, the spacer comprises a sequence as set forth in SEQ ID NO: 22 or SEQ ID NO: 23. In some of the embodiments, the spacer is set forth in SEQ ID NO: 22.

[0018] In some of the embodiments, the spacer is 100-150 amino acids in length. In some of the embodiments, the spacer is 110-130 amino acids in length. In some of the embodiments, the spacer comprises an immunoglobulin hinge region and an immunoglobulin CH3 region. In some of the embodiments, the spacer comprises an IgG4 hinge region or a variant thereof and an IgG4 CH3 region. In some of the embodiments, the spacer comprises the sequence set forth in SEQ ID NO:24 or SEQ ID NO:96. In some of the embodiments, the spacer is set forth in SEQ ID NO:24.

[0019] In some of the embodiments, the spacer is 200-250 amino acids in length. In some of the embodiments, the spacer is 220-240 amino acids in length. In some of the embodiments, the spacer comprises an immunoglobulin hinge region, an immunoglobulin CH2 region or a chimeric CH2 region of two different immunoglobulins, and an immunoglobulin CH3 region. In some of the embodiments, the spacer comprises an IgG4 hinge region or a variant thereof, a chimeric CH2 region (IgG2 / 4 CH2 region) comprising a portion of an IgG4 CH2 and a portion of an IgG2 CH2, and an IgG4 CH3 region. In some of the embodiments, the spacer comprises a sequence as set forth in SEQ ID NO:25 or SEQ ID NO:97. In some of the embodiments, the spacer is set forth in SEQ ID NO:25. In some of the embodiments, the spacer comprises the amino acids GGGGS (SEQ ID NO:99). In some of the embodiments, the spacer is set forth in SEQ ID NO:99. In some of the embodiments, the spacer is set forth in SEQ ID NO:100.

[0020] In some of any of the embodiments, the CAAR comprises a sequence of amino acids that exhibits at least 85% sequence identity to any of the sequences set forth in SEQ ID NO: 29, 30, 31, 38, 39, 40, 53, 54, 55, 56, 57, 58, 104, 110, or 111. In some embodiments, the CAAR comprises a sequence set forth in any one of SEQ ID NO: 29, 30, 31, 38, 39, 40, 53, 54, 55, 56, 57, 58, 104, 110, or 111. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 29, or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO: 29. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 30, or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO: 30. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 38, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 38. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 55, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 55. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 56, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 56. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 57, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 57. Provided herein is a CAAR comprising a sequence of amino acids set forth in SEQ ID NO: 58, or a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 58.

[0021] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain comprising the sequence shown in SEQ ID NO:9; (b) a spacer comprising the sequence shown in SEQ ID NO:25; (c) a transmembrane region comprising the sequence shown in SEQ ID NO:26; (d) a costimulatory signaling region comprising the sequence shown in SEQ ID NO:27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO:28. Provided herein is a CAAR comprising an amino acid sequence that exhibits at least 90% sequence identity with SEQ ID NO:55, and the amino acid sequence comprises a G4P mutation, based on the numbering of positions in SEQ ID NO:1. In some embodiments, the CAAR comprises an amino acid sequence shown in SEQ ID NO:55.

[0022] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain comprising the sequence shown in SEQ ID NO: 10; (b) a spacer comprising the sequence shown in SEQ ID NO: 24; (c) a transmembrane region comprising the sequence shown in SEQ ID NO: 26; (d) a costimulatory signaling region comprising the sequence shown in SEQ ID NO: 27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO: 28. Provided herein is a CAAR comprising an amino acid sequence that exhibits at least 90% sequence identity with SEQ ID NO: 57, and the amino acid sequence comprises a G4P mutation, based on the numbering of positions in SEQ ID NO: 1. In some embodiments, the CAAR comprises an amino acid sequence shown in SEQ ID NO: 57.

[0023] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain comprising the sequence shown in SEQ ID NO:9; (b) a spacer comprising the sequence shown in SEQ ID NO:100; (c) a transmembrane region comprising the sequence shown in SEQ ID NO:26; (d) a costimulatory signaling region comprising the sequence shown in SEQ ID NO:27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO:28. Provided herein is a CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO:104, and the sequence of amino acids comprises a G4P mutation, based on the numbering of positions in SEQ ID NO:1. In some embodiments, the CAAR comprises a sequence of amino acids as shown in SEQ ID NO:104.

[0024] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain comprising the sequence shown in SEQ ID NO:9; (b) a spacer comprising the sequence shown in SEQ ID NO:22; (c) a transmembrane region comprising the sequence shown in SEQ ID NO:98; (d) a costimulatory signaling region comprising the sequence shown in SEQ ID NO:27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO:28. Provided herein is a CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO:110, and the sequence of amino acids comprises a G4P mutation, based on the numbering of positions in SEQ ID NO:1. In some embodiments, the CAAR comprises a sequence of amino acids as shown in SEQ ID NO:110.

[0025] Provided herein is a CAAR comprising: (a) a PR3 antibody binding domain comprising the sequence shown in SEQ ID NO: 10; (b) a spacer comprising the sequence shown in SEQ ID NO: 100; (c) a transmembrane region comprising the sequence shown in SEQ ID NO: 98; (d) a costimulatory signaling region comprising the sequence shown in SEQ ID NO: 27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO: 28. Provided herein is a CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 111 or 119, and the sequence of amino acids comprises H44A mutation, based on the numbering of positions in SEQ ID NO: 1. In some embodiments, the CAAR comprises a sequence of amino acids as shown in SEQ ID NO: 111 or 119.

[0026] In some of any of the embodiments, CAAR can be specifically bound by anti-PR3 anti-neutrophil cytoplasmic antibody (PR3-ANCA). In some of any of the embodiments, the PR3 antibody binding domain comprises at least 2, 3, 4, 5, 6 or 7 epitopes recognized by anti-PR3 anti-neutrophil cytoplasmic antibody (PR3-ANCA). In some of the embodiments, the epitopes are selected from the group consisting of ANCA2 (AQPHSRPYMAS, SEQ ID NO: 90), ANCA3 (PGSHFCGG, SEQ ID NO: 91), ANCA4 (VVLGAHNVRTQ, SEQ ID NO: 92), ANCA5 (FLNNYDAE, SEQ ID NO: 93), ANCA6 (PVPHGTQC, SEQ ID NO: 94) and ANCA7 (CFGDSGGP, SEQ ID NO: 95).

[0027] In some of the embodiments, the CAAR is specific for PR3-reactive B cells. In some of the embodiments, Jurkat reporter cells transduced with CAAR, having Td-tomato knocked into the endogenous Nur77 locus, show tonic signaling of less than or equal to 25%, 20%, 15%, 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2%, or 1% as measured by Td-tomato fluorescence by flow cytometry and determined as a total percentage of cells expressing Td-tomato in the transduced Jurkat reporter cells. In some of the embodiments, the cells transduced with CAAR show antigen-specific activation after exposure to anti-human PR3 antibody. In some of any of the embodiments, the CAAR comprises a sequence of amino acids that exhibits at least 85% sequence identity to any of the sequences set forth in SEQ ID NOs: 55, 57, 104, 110, and 111. In some of any of the embodiments, the CAAR comprises a sequence set forth in any one of SEQ ID NOs: 55, 57, 104, 110, and 111.

[0028] In some of any of the embodiments, the CAAR can kill at least 65% of anti-PR3 antibody expressing cellular targets. In some of any of the embodiments, the CAAR can kill at least 65% of each of at least three cellular targets expressing different anti-PR3 antibodies, and each of the at least three different antibodies binds to a different PR3 epitope. In some of any of the embodiments, the CAAR comprises a sequence of amino acids that exhibits at least 85% sequence identity with any of the sequences shown in SEQ ID NOs: 55, 57, 104, 110, and 111. In some of any of the embodiments, the CAAR comprises a sequence shown in any one of SEQ ID NOs: 55, 57, 104, 110, and 111.

[0029] In some of the embodiments, the polynucleotide comprises the nucleic acid encoding CAAR.In some of the embodiments, the polynucleotide is optimized by splice site elimination.In some of the embodiments, the polynucleotide is codon-optimized for expression in human cells.In some of the embodiments, the polynucleotide comprises the nucleic acid sequence that shows at least 85% sequence identity with the sequence shown in any of SEQ ID NO: 59, 60, 61, 68, 69, 70, 83, 84, 85, 86, 87, 88, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 or 159. In some embodiments, the polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 59, 60, 61, 68, 69, 70, 83, 84, 85, 86, 87, 88, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, or 159. In some of any of the embodiments, the polynucleotide comprises a nucleic acid sequence that exhibits at least 90% sequence identity to a sequence set forth in any of SEQ ID NOs: 59, 60, 68, 85, 86, 87, 88, 145, 151, or 152. In some embodiments, the polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 59, 60, 68, 85, 86, 87, or 88.

[0030] In some of the embodiments, the vector comprises a polynucleotide. In some of the embodiments, the vector is a viral vector. In some of the embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector). In some of the embodiments, the cell comprises a CAAR. In some of the embodiments, the cell comprises a polynucleotide or a vector. In some of the embodiments, the cell is a lymphocyte. In some of the embodiments, the cell is a NK cell or a T cell. In some of the embodiments, the cell is a T cell, and the T cell is a CD4+ T cell or a CD8+ T cell. In some of the embodiments, the cell is a primary cell obtained from a subject. In some of the embodiments, the cell is an induced pluripotent stem cell. In some of the embodiments, the cell is differentiated from an induced pluripotent stem cell. In some of the embodiments, the cell is an allogeneic cell. In some of the embodiments, the cell is engineered to be hypoimmune.

[0031] In some of the embodiments, the cells exhibit cytotoxic activity against PR3-reactive B cells. In some of the embodiments, the cytotoxic activity is against a plurality of PR3-reactive B cells, and the plurality of PR3-reactive B cells are specific for two or more different PR3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 2, 3, 4, 5, 6 or 7 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 4 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 5 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 6 different PR3 ANCA epitopes. In some of any of the embodiments, the ANCA epitope is selected from the group consisting of ANCA2 (AQPHSRPYMAS, SEQ ID NO: 90), ANCA3 (PGSHFCGG, SEQ ID NO: 91), ANCA4 (VVLGAHNVRTQ, SEQ ID NO: 92), ANCA5 (FLNNYDAE, SEQ ID NO: 93), ANCA6 (PVPHGTQC, SEQ ID NO: 94) and ANCA7 (CFGDSGGP, SEQ ID NO: 95).

[0032] In some of the embodiments, the composition comprises cells. In some of the embodiments, the composition further comprises a pharma- ceutically acceptable excipient. In some of the embodiments, the composition comprises CD4+ and CD8+ T cells. In some of the embodiments, the ratio of CD4+ T cells to CD8+ T cells is 1:3 to 3:1 or about 1:3 to 3:1. In some of the embodiments, the ratio of CD4+ T cells to CD8+ T cells is 1:2 to 2:1 or about 1:2 to 2:1. In some of the embodiments, the ratio of CD4+ to CD8+ is 1:1 or about 1:1. In some of the embodiments, at least 90% or more than 90%, at least 95% or more than 95%, or at least 98% or more than 98% of the cells in the composition are CD3+ T cells. In some of the embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the composition express CAAR. In some of any of the embodiments, among the plurality of cells in the composition that express CAAR, 10% or about 10%, 9% or about 9%, 8% or about 8%, 7% or about 7%, 5% or about 5%, 4% or about 4%, 3% or about 3%, 2% or about 2%, or less than 1% or about 1% of the cells in the plurality of cells exhibit tonic signaling and / or antigen-independent activity or signaling. In some of any of the embodiments, the method for killing PR3-reactive B cells comprises contacting PR3-reactive B cells with a cell or a composition. In some of the embodiments, the method is performed in vitro or ex vivo. In some of the embodiments, the method is performed in vivo in a subject.

[0033] In some of the embodiments, the PR3-reactive B cells comprise a plurality of PR3-reactive B cells, and the plurality of PR3-reactive B cells are specific for two or more different PR3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for 2, 3, 4, 5, 6 or 7 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 4 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 5 different PR3 ANCA epitopes. In some of the embodiments, the plurality of PR3-reactive B cells are specific for at least 6 different PR3 ANCA epitopes. In some of any of the embodiments, the ANCA epitope is selected from the group consisting of ANCA2 (AQPHSRPYMAS, SEQ ID NO: 90), ANCA3 (PGSHFCGG, SEQ ID NO: 91), ANCA4 (VVLGAHNVRTQ, SEQ ID NO: 92), ANCA5 (FLNNYDAE, SEQ ID NO: 93), ANCA6 (PVPHGTQC, SEQ ID NO: 94) and ANCA7 (CFGDSGGP, SEQ ID NO: 95).

[0034] In some of the embodiments, among the plurality of PR3-reactive B cells, the method results in killing of at or above about 80%, at or above about 90%, at or above about 90%, at or above about 95% of the PR3-reactive B cells. In some of the embodiments, the PR3-reactive B cells are IgG+. In some of the embodiments, the method includes administering the cells or the composition to a subject in need of such treatment. In some of the embodiments, the disease or disorder is an autoimmune condition. In some of the embodiments, the disease or disorder is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In some of the embodiments, the AAV is PR3-AAV. In some of the embodiments, the disease or disorder is selected from the group consisting of granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), or eosinophilic granulomatosis with polyangiitis (EGPA). In some of the embodiments, the subject is positive for PR3-ANCA. In some of the optional embodiments, the method includes testing the subject for PR3-ANCA. In some embodiments, the method includes verifying that the subject has tested positive for PR3-ANCA.

[0035] In some of the embodiments, the subject has been treated with a previous treatment (e.g., one or more previous treatments) against AAV. In some of the embodiments, the subject has not achieved remission or has relapsed after treatment with a previous treatment. In some of the embodiments, the previous treatment includes one or more of glucocorticoids, cyclophosphamide (CYC), rituximab (RTX), plasma exchange, avacopan, methotrexate (MTX), mycophenolate mofetil (MMF), azathioprine (AZA), leflunomide (LEF), belimumab, mepolizumab, and omalizumab. In some of the embodiments, the glucocorticoid is one or more of prednisolone, methylprednisolone, prednisone, and dexamethasone.

[0036] In some of any of the embodiments, the previous treatment comprises a remission induction treatment (e.g., a remission induction agent). In some embodiments, the remission induction treatment comprises one or more of rituximab, cyclophosphamide, and a glucocorticoid. In some embodiments, the glucocorticoid is one or more of prednisolone, methylprednisolone, prednisone, and dexamethasone. In some embodiments, the glucocorticoid is prednisolone.

[0037] In some of the embodiments, the previous treatment comprises a remission maintenance treatment. In some of the embodiments, the remission maintenance treatment comprises one or more of rituximab, methotrexate, azathioprine, mycophenolate mofetil, leflunomide, mepolizumab, and omalizumab. In some of the embodiments, the method of treating a subject with PR3-ANCA vasculitis comprises (i) administering a remission induction treatment to the subject, and (ii) administering a cell or composition to the subject. In some of the embodiments, the remission induction treatment comprises cyclophosphamide. In some of the embodiments, the cyclophosphamide is administered to the subject prior to administering the cell or composition to the subject. In some of the embodiments, the course of cyclophosphamide is administered to the subject at least 1 week, 2 weeks, 3 weeks, or 4 weeks prior to administering the cell or composition to the subject. In some of the embodiments, the remission induction treatment further comprises a glucocorticoid. In some of the embodiments, the glucocorticoid is prednisolone.

[0038] In some of the embodiments, the method for treating a subject having PR3-ANCA vasculitis includes (i) administering cyclophosphamide to the subject, and (ii) administering a cell or a composition to the subject. In some of the embodiments, the method includes administering cyclophosphamide to the subject before administering the cell or composition to the subject. In some of the embodiments, the course of cyclophosphamide is administered to the subject at least 1, 2, 3, or 4 weeks before administering the cell or composition to the subject. In some of the embodiments, the course of cyclophosphamide is administered according to a dosing schedule determined to be sufficient to reduce ANCA titers in patients having PR3-ANCA vasculitis. In some of the embodiments, the cyclophosphamide is administered at an oral dose of 2 mg / kg / day at least 1, 2, 3, or 4 weeks before administering the cell or composition to the subject. In some of the embodiments, the cyclophosphamide is administered at an IV dose of 15 mg / kg every 2 weeks for up to 3 doses before administering the cell or composition to the subject. In some of any of the embodiments, after administering the cells or compositions to the subject, the subject is not treated with cyclophosphamide.

[0039] In some of the embodiments, the method comprises administering a glucocorticoid to the subject before administering the cell or composition to the subject. In some of the embodiments, the method comprises administering a glucocorticoid to the subject after administering the cell or composition to the subject. In some of the embodiments, the method for treating a subject with PR3-ANCA vasculitis comprises (i) administering a preconditioning therapy to the subject that has been shown to be effective in depleting ANCA, followed by (ii) administering the cell or composition to the subject. In some of the embodiments, the preconditioning therapy comprises cyclophosphamide. [Brief description of the drawings]

[0040] [Figure 1]FIG. 1 shows a schematic diagram of the PR3 CAAR construct showing components including the signal peptide, the extracellular anti-PR3 antibody binding domain (PR3), the spacer, the transmembrane domain (TM), the costimulatory (Co-Stim) domain and the signaling domain. [Figure 2-1] FIG. 2 shows the differential tonic signaling and activation observed across the various PR3-CAAR-expressing Nur77-Tdtomato reporter Jurkat T cells described in Example 1 after 24 hours of incubation in the absence of target (PBS control condition, top row) and in the presence of targeting α-PR3 (anti-PR3) antibody (activating condition, bottom row). [Figure 2-2] Same as above. [Diagram 3] Figure 3 shows the reported PR3 ANCA epitopes on the PR3 crystal structure (left) and epitope binning (right) of PR3-reactive antibodies generated through subcutaneous immunization of rodents with native active human PR3 protein compared to a reference antibody. Each circle in the cluster plot represents an antibody in a pairwise competition experiment, and the lines indicate the competition between the two antibodies connected by a line. Antibodies are grouped into epitope bins as shown based on similar blocking profiles. [Figure 4] Figure 4 shows the results from an in vitro cytotoxicity assay performed to evaluate the impact of target to effector ratios, as described in Example 4, part A. Killing efficiency was evaluated by measuring the loss of viable target cells at 24 hours after co-culture of IgG+ CHO-S target cells and primary T cells expressing one of the four PR3 CAARs or no CAR (mock CAAR). As a control, culture of mock CAR cells (T cells not expressing a CAR) with target cells was evaluated. [Diagram 5] FIG. 5 shows CAAR-T cell potency against targets expressing different anti-PR3 antibodies with various epitope reactivities, as described in Example 4, part B. [Figure 6]Figure 6 shows the effect of anti-PR3 antibody surface density on PR3-CAAR T cell potency. αPR3 antibody expression on IgG+-CHO-S targets was assessed by mean fluorescence intensity (MFI), and CAAR T cell killing was assessed against individual IgG+-CHO-S targets. Killing efficiency against high density targets (H) and low density targets (L), as well as against combined high and low density targets (pool) are shown. [Figure 7] Figure 7 shows CAAR T cell activation in the absence of target cells (CAAR alone) and after 24 hours of exposure to anti-PR3 expressing cell targets (L=low density, H=high density, pool=combination of low and high density). Activation was measured by CD69 levels on CD8+ PR3-CAAR T cells. [Figure 8] FIG. 8 shows the killing efficiency of PR3 CAAR T cells in the presence of soluble anti-PR3 monoclonal antibodies (left graph) or soluble antineutrophil cytoplasmic autoantibodies (ANCA) (right graph) derived from a patient with ANCA-associated vasculitis. [Figure 9-1]Figures 9A-9C show in vivo PR3-CAAR T cell targeting of immune-induced PR3-reactive B cell populations. Figure 9A (left panel) shows that PR3-immunized mice receiving PR3-CAAR T cells showed statistically significant depletion of PR3-reactive splenic class-switched B2 cell populations. The top left FACS dot plot shows the presence of PR3-reactive class-switched B2 cells after treatment with mock CAAR T cells, and the right graph shows depletion of such cells after treatment with PR3 CAAR T cells. The bottom graph shows that the percentage of PR3-reactive splenic class-switched B2 cells after treatment with PR3 CAAR T cells was significantly reduced compared to control conditions (mock CAAR T cells and no T cells). Figure 9B (middle panel) shows that PR3-immunized mice receiving PR3-CAAR T cells showed a statistically significant depletion of PR3-reactive CD19-CD43+ cells in the bone marrow, as shown by the percentage (top graph) and total counts (bottom graph) of such cells. Figure 9C (right panel) shows that PR3-immunized mice receiving PR3-CAAR T cells showed a statistically significant depletion of PR3-reactive class-switched B2 cells in the mesenteric lymph nodes, as shown by the percentage (top graph) and total counts (bottom graph) of such cells. [Figure 9-2] Same as above. [Figure 10] FIG. 10 shows minimal off-target killing of IgGneg hybridomas by PR3-CAAR T cells with G4S linker. [Figure 11] Figure 11 shows CAAR T cell activation in the absence of target cells (CAR only) and after 24 hours of exposure to anti-PR3 expressing cell targets. Activation was measured by CD69 levels on CD8+ PR3-CAAR T cells. [Figure 12] FIG. 12 shows in vivo PR3-CAAR T cell targeting of PR3-reactive K562 cells in an NSG xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Provided herein is a cell or biological (e.g., protein) therapeutic comprising a PR3 antibody binding domain, such as a wild-type or mutant PR3 protein. The term "PR3 antibody binding domain" as used herein is a protein or polypeptide that can receive binding of one or more anti-PR3 antibodies (e.g., one or more PR3 ANCAs). The PR3 antibody binding domain can be a wild-type or mutant PR3 protein, or a portion of a wild-type or mutant PR3 protein. In some embodiments, the PR3 antibody binding domain comprises one or more ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises multiple ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least 2, 3, 4, 5, 6, or 7 ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least 1, 2, 3, 4, 5, 6, or 7 ANCA epitopes disclosed in Table 1. In some embodiments, the PR3 antibody binding domain comprises multiple (e.g., at least 2, 3, 4, 5, 6, or 7) ANCA epitopes disclosed in Table 1. In some embodiments, the PR3 antibody binding domain is one or more of the PR3 antibody binding domains described in Section IA.

[0042] In certain embodiments, the PR3 antibody binding domain, e.g., a mutant PR3 protein, has a mutation (e.g., a point mutation) compared to a wild-type PR3 protein. In some embodiments, the mutation of the PR3 antibody binding domain reduces its enzymatic activity (compared to the corresponding binding domain that does not contain the mutation) and / or the mutation renders the PR3 antibody binding domain enzymatically inactive. In some embodiments, the corresponding antibody binding domain is a wild-type PR3 protein (e.g., a wild-type PR3 protein having the sequence of SEQ ID NO: 1). The enzymatic activity of the PR3 antibody binding domain can be assessed using the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (MeAAPV) (MilliporeSigma) according to Silva et al. (2010) J Autoimmunity and Van Der Geld et al. (2002) Clin Exp Immunol. In some embodiments, the proteolysis of a synthetic substrate by the PR3 antibody binding domain is monitored via measuring absorbance at 405 nm over time. In some embodiments, specific activity can be calculated by reference to a standard with p-nitroaniline (truncated substrate). In certain embodiments, the PR3 antibody binding domain, e.g., a mutant PR3 protein, contains one or more epitopes recognized by or bound by PR3-ANCA.

[0043] In some embodiments, the biological (e.g., protein) therapeutic comprises a soluble PR3 antibody binding domain (e.g., a wild-type or mutant PR3 protein, or a portion of a wild-type or mutant PR3 protein). In some embodiments, the cellular therapeutic comprises a cell and a PR3 antibody binding domain (e.g., a wild-type or mutant PR3 protein) expressed on the surface of the cell. In some embodiments, the PR3 antibody binding domain is part of a recombinant receptor.

[0044] Also provided herein are methods of treating autoimmune diseases (e.g., AAV) comprising administering a cell or protein therapeutic.

[0045] In another aspect, provided herein is a method of depleting antigen-specific immune cells, the method comprising administering a cell or biological therapeutic (e.g., a protein) comprising a mutant PR3 protein that binds or can bind to at least one, two, three, four, five, six, or seven epitopes on PR3-specific immune cells (e.g., at least one, two, three, four, five, six, or seven epitopes of ANCA disclosed in Table 1). In one embodiment, the therapeutic is a cell therapeutic. In another embodiment, the therapeutic is a protein therapeutic.

[0046] Among the embodiments provided are cellular therapeutics that contain wild-type or mutant PR3 proteins. In some embodiments, a chimeric autoantibody receptor (CAAR) is provided herein that includes an extracellular proteinase 3 (PR3) antibody binding domain. In some embodiments, the extracellular domain of the provided CAAR consists of a PR3 antibody binding domain. In some embodiments, the PR3 antibody binding domain can receive binding from anti-neutrophil cytoplasmic antibodies (ANCA), and thus serves as a binding target for recognition by disease-causing autoantibodies. Thus, autoantibodies expressed on autoreactive cells serve as specific targets for CAAR, which can then destroy or kill the autoreactive cells. Also provided herein are methods for CAAR to target autoantibodies and the B cells that generate them. In some embodiments, such methods include methods of treatment and use for treating diseases or conditions, such as autoimmune diseases or conditions, such as anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), which are typically caused by ANCA that targets PR3.

[0047] PR3, expressed in granulomatous lesions, is an autoantigen target for autoreactive B and T cells. Autoantigens can include autoantigens or antigens from normal tissues that are cell-mediated targets, or antibody-mediated immune responses that can lead to the development of autoimmune diseases. The autoantigen PR3 (neutrophil serine protease proteinase 3) can be bound by antineutrophil cytoplasmic antibodies (ANCA), which induce neutrophils to generate superoxide and alter their apoptosis. Anti-PR3 autoantibodies (PR3-ANCA) target and bind PR3 and are associated with the development of vasculitis.

[0048] Certain therapeutic interventions for treating such autoantibody-mediated diseases target immune cells, such as B cells, that produce and present autoantibodies on their cell surface. The advantage of the approach disclosed herein is that it allows targeted killing of immune cells that express anti-PR3 autoantibodies, such as B cells that express anti-PR3 autoantibodies. It is expected that other immune cells (including other B cells) are not targeted by this approach, and therefore they can continue to produce antibodies that protect the subject from infection. Furthermore, among the CAARs provided are those in which the PR3 antibody binding domain is recognized by multiple different ANCAs (e.g., 2, 3, 4, 5, 6 or more), providing a breadth of killing activity against cells that express different reactive autoantibodies. The CAARs provided also show hypotonic (as exemplified herein, hypotonic activity) antibody target specific killing.

[0049] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. In the event that a definition set forth herein is opposed to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0051] I.PR3 Chimeric Autoantibody Receptor In some aspects, CAAR is provided that comprises PR3 antibody binding domain. Typically, PR3 antibody binding domain can be specifically bound by anti-PR3 antibody, for example, one or more PR3 ANCA. Also provided is a polynucleotide that encodes PR3 CAAR. In some embodiments, the polynucleotide provided can be incorporated into DNA or RNA constructs, such as those that can be introduced into cells. Also provided herein are cells that express PR3 CAAR, compositions that contain such cells, and their use in adoptive cell therapy.

[0052] A. PR3 antibody binding domain The CAAR disclosed herein generally comprises an extracellular PR3 antibody binding domain (e.g., a PR3 autoantibody binding domain). The PR3 antibody binding domain may be a wild-type PR3 protein or a fragment or mutant thereof. In certain embodiments, the antibody binding domain comprises a PR3 protein or a fragment or mutant thereof that can be specifically bound by anti-PR3 antineutrophil cytoplasmic antibody (PR3-ANCA). The extracellular antibody binding domain of the provided CAAR comprises one or more epitopes that are recognized or bound by PR3-ANCA. In some embodiments, the extracellular antibody binding domain is a PR3 autoantigen.

[0053] The serine protease proteinase 3 (PR3) is an enzyme released during inflammation in neutrophils. PR3 is initially transcribed as an inactive precursor (zymogen) and then undergoes two steps of post-translational modification to become the active form. First, there is cleavage of the N-terminal signal peptide (via a signal peptidase), followed by cleavage of the N-terminal prodipeptide by the cysteine ​​proteinase, cathepsin C, which is essential for enzymatic activity. Second, the propeptide form undergoes propeptide cleavage at the C-terminus, which is crucial for granule packaging. This forms the catalytic triad of residues and the final conformation of mature PR3 (Crisford et al., Respiratory Research, volume 19, Article number: 180, 2018). Residues histidine-44, aspartic acid-91, and serine-176 are the triad that constitute the active site of PR3.

[0054] In some embodiments, the PR3 antibody binding domain is a wild-type PR3 protein. In some embodiments, the PR3 protein is a human protein. In some embodiments, the PR3 antibody binding domain is a precursor PR3 protein, such as PR3 with an N-terminal dipeptide and / or a C-terminal propeptide. In some embodiments, the PR3 antibody binding domain is a mature PR3 protein. The PR3 antibody binding domain can also be a mutant or isoform of human PR3, including a variant of human PR3, such as a precursor or mature PR3 of human PR3.

[0055] In some embodiments, the PR3 antibody binding domain is a precursor PR3 protein that is not processed at the N-terminus and / or C-terminus. In some embodiments, the precursor PR3 protein is not catalytically active. In some embodiments, the PR3 protein contains an N-terminal dipeptide. In some embodiments, the PR3 antibody binding domain comprises the sequence of SEQ ID NO:5. In some embodiments, the PR3 protein contains an N-terminal dipeptide. In some embodiments, the PR3 antibody binding domain comprises the sequence of SEQ ID NO:3. The PR3 antibody binding domain can also be a variant of any of the foregoing, such as a mutant or isoform of any of the foregoing. In some embodiments, the PR3 antibody binding domain has a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or 5.

[0056] In some embodiments, the PR3 antibody binding domain is a mature wild-type PR3 protein. In some embodiments, the PR3 antibody binding domain comprises the sequence of SEQ ID NO:1. In some embodiments, the PR3 antibody binding domain is a mature PR3 protein having the sequence of SEQ ID NO:1. The PR3 antibody binding domain can also be a variant of any of the foregoing, such as a mutant or isoform of SEQ ID NO:1. In some embodiments, the PR3 antibody binding domain has a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0057] In some embodiments, the PR3 antibody binding domain is a mutant PR3 protein. In certain embodiments, the PR3 antibody binding domain is a mutant PR3 protein having a mutation (e.g., a point mutation) compared to wild-type PR3, e.g., compared to the PR3 shown in SEQ ID NO:1. The point mutation can occur, for example, at a residue corresponding to residue 4, 44, 91, 175, or 176 of SEQ ID NO:1. In some embodiments, the point mutation can be to any other amino acid at that position. In some embodiments, the point mutation is to any other amino acid other than an amino acid with a bulky side chain. In some embodiments, the point mutation is to alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), glycine (G), proline (P), cysteine ​​(C), serine (S), threonine (T), asparagine (N), or glutamine (Q). In some embodiments, the amino acid mutation is G4P, H44A, D91N, D175N, S176A or S176C.

[0058] In some embodiments, the point mutation in PR3 reduces the enzymatic activity of PR3, such as rendering the antibody binding domain enzymatically inactive. In some embodiments, the point mutation in PR3 reduces the enzymatic activity of the mutant PR3 compared to a wild-type PR3 (e.g., a wild-type PR3 comprising the sequence of SEQ ID NO:1). In some embodiments, the point mutation in PR3 reduces the enzymatic activity of the mutant PR3 by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the enzymatic activity of the wild-type PR3 (e.g., as set forth in SEQ ID NO:1). In certain embodiments, the point mutation in PR3 reduces the enzymatic activity of the mutant PR3 by at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. In some embodiments, the point mutation in PR3 reduces the enzymatic activity by 1% to 11%, 10% to 21%, 20% to 31%, 30% to 41%, 40% to 51%, 50% to 61%, 60% to 71%, 70% to 81%, 80% to 91%, or 90% to 100% compared to the enzymatic activity of wild-type PR3 (e.g., as shown in SEQ ID NO:1). In certain embodiments, the enzymatic activity is reduced by at least 50%, e.g., typically at least 60%, 70%, 80%, 90% or more compared to the enzymatic activity of wild-type PR3 (e.g., as shown in SEQ ID NO:1).

[0059] The PR3 antibody binding domain of CAAR provided herein can be recognized and bound by anti-PR3 antibodies, such as PR3 anti-neutrophil cytoplasmic antibodies (PR3-ANCA). ANCA is found in the serum of about 90% of all active and / or untreated Wegener's granulomatosis (WG) patients, and about 90% of all cytoplasmic ANCA WG patients bind PR3 (Bruner et al. (2010) Clin and Experimental Immunol). In some embodiments, the PR3 antibody binding domain disclosed herein comprises one or more ANCA binding epitopes. In some embodiments, the PR3 antibody binding domain comprises 1, 2, 3, 4, 5, 6, or 7 ANCA epitopes listed in Table 1 below.

[0060] [Table 1]

[0061] In some embodiments, the PR3 antibody binding domain contains one or more PR3 epitopes that can be subjected to ANCA binding. FIG. 3 illustrates certain ANCA epitopes that can be present on the PR3 antibody binding domain of the provided CAAR. In some embodiments, any one or more, e.g., a combination of two or more, of the ANCA epitopes in FIG. 3 can be present on the PR3 antibody binding domain of the provided CAAR. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 2. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 3. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 4. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 5. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 6. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 7. In some embodiments, the PR3 antibody binding domain comprises ANCA epitope 1. In some embodiments, the PR3 antibody binding domain comprises a combination of two ANCA epitopes, three ANCA epitopes, four ANCA epitopes, five ANCA epitopes, six ANCA epitopes, or seven ANCA epitopes.

[0062] In some embodiments, the PR3 antibody binding domain comprises at least two ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least three ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least four ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least five ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises five ANCA epitopes. In some embodiments, the PR3 antibody binding domain comprises at least six ANCA epitopes. In some aspects of the foregoing embodiments, the ANCA epitopes comprise one or more of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95. In some aspects of the foregoing embodiments, the ANCA epitopes comprise one or more of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95. In some aspects of the foregoing embodiments, the ANCA epitope is selected from SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95. In some aspects of the foregoing embodiments, the ANCA epitope is selected from SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95.

[0063] In some embodiments, the PR3 antibody binding domain comprises at least two, three, four, five or six of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the PR3 antibody binding domain comprises in its sequence at least two epitope sequences selected from the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the PR3 antibody binding domain comprises in its sequence at least three epitope sequences from the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the PR3 antibody binding domain comprises in its sequence at least four epitope sequences selected from the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95. In some embodiments, the PR3 antibody binding domain comprises in its sequence at least five epitope sequences selected from the group consisting of SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95. In some embodiments, the PR3 antibody binding domain comprises in its sequence the epitope sequences set forth as SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95.

[0064] In certain embodiments, the human PR3 antibody binding domain has a length of 231 amino acids or less. In certain embodiments, the human PR3 antibody binding domain has a length of 229 amino acids or less, 227 amino acids or less, 225 amino acids or less, 223 amino acids or less, 222 amino acids or less, 221 amino acids or less, 219 amino acids or less, or 217 amino acids or less. In some embodiments, the PR3 antibody binding domain has a length of 217-231 amino acids. In some embodiments, the PR3 antibody binding domain has a length of 217-222 amino acids. In some embodiments, the PR3 antibody binding domain has a length of 219-231 amino acids. In some embodiments, the PR3 antibody binding domain has a length of 221-231 amino acids. In some embodiments, the PR3 antibody binding domain has a length of 221 amino acids. In some embodiments, the PR3 antibody binding domain has a length of 221 amino acids.

[0065] In some embodiments, the CAAR is specific for PR3-reactive immune cells (e.g., B cells). In some embodiments, "specific" in terms of binding refers to the binding of a protein (e.g., a ligand) to a cognate binding partner protein present in a sample or expressed on a cell, but the protein does not substantially recognize or bind to other molecules in the sample or expressed on the cell. For example, an extracellular PR3 antibody binding domain is specific for PR3-ANCA expressed on one or more reactive immune cells, such as B cells. In some embodiments, the observation that a protein or other binding molecule binds to a particular PR3-ANCA or specifically binds to a PR3-ANCA protein does not necessarily mean that it binds to a different PR3-ANCA that recognizes a different epitope.

[0066] In some embodiments, the extent of binding of the PR3 antibody binding domain or CAAR containing it to an unrelated, non-PR3-ANCA protein is less than or about 10% of the binding of the PR3 antibody binding domain or CAAR to the target PR3-ANCA, as measured, for example, by flow cytometry. A variety of assays are known for assessing binding or for determining whether a protein (e.g., a ligand) specifically binds to its cognate binding partner (e.g., PR3-ANCA). Suitable assays for measuring binding of one protein to another include, for example, using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993, and U.S. Pat. Nos. 5,283,173 and 5,468,614; or equivalents), immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins via fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detecting binding of expressed polynucleotides or proteins. In particular, binding can be assessed by flow cytometry.

[0067] 1. Exemplary PR3 Antibody Binding Domains A PR3 CAAR generally comprises an extracellular antibody binding domain (e.g., an extracellular autoantibody binding domain) that includes, is, or comprises one or more of the PR3 antibody binding domains disclosed herein. In some of the provided embodiments, the PR3 antibody binding domain is or comprises a sequence set forth in any one of SEQ ID NOs: 1-10, or is or comprises a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1-10.

[0068] In some embodiments, the PR3 antibody binding domain is a mature human PR3 antibody binding domain, e.g., as set forth in SEQ ID NO: 1. In some embodiments, the PR3 antibody binding domain comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the PR3 antibody binding domain consists of the sequence set forth in SEQ ID NO: 1.

[0069] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 176 compared to mature human PR3, e.g., mature human PR3 as set forth in SEQ ID NO: 1. In some embodiments, the mutation is S176A. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence as set forth in SEQ ID NO: 2 and contains the mutation S176A. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:2.

[0070] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain that contains an unprocessed C-terminus compared to mature human PR3, e.g., mature human PR3 as set forth in SEQ ID NO: 1. In some embodiments, the PR3 antibody binding domain comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, or 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 and contains an unprocessed C-terminus. In some embodiments, the PR3 antibody binding domain consists of the sequence set forth in SEQ ID NO: 3.

[0071] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 91 compared to mature human PR3, e.g., mature human PR3 set forth in SEQ ID NO: 1. In some embodiments, the mutation is D91N. In some embodiments, the PR3 antibody binding domain comprises the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence set forth in SEQ ID NO: 4 and contains the mutation D91N. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:4.

[0072] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain that contains an unprocessed N-terminus compared to mature human PR3, e.g., mature human PR3 as set forth in SEQ ID NO: 1. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence as set forth in SEQ ID NO: 5 and contains an unprocessed N-terminus. In some embodiments, the PR3 antibody binding domain consists of the sequence as set forth in SEQ ID NO: 5.

[0073] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at the N-terminus compared to mature human PR3, e.g., mature human PR3 set forth in SEQ ID NO: 1. In some embodiments, the mutation is an N-terminal Ile deletion. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence set forth in SEQ ID NO: 6 and contains an N-terminal Ile deletion. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:6.

[0074] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 175 compared to mature human PR3, e.g., mature human PR3 set forth in SEQ ID NO: 1. In some embodiments, the mutation is D175N. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence set forth in SEQ ID NO: 7 and contains the mutation D175N. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:7.

[0075] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 176 compared to mature human PR3, e.g., mature human PR3 set forth in SEQ ID NO: 1. In some embodiments, the mutation is S176C. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence set forth in SEQ ID NO: 8 and contains the mutation S176C. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:8.

[0076] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 4 compared to mature human PR3, e.g., mature human PR3 as set forth in SEQ ID NO:1. In some embodiments, the mutation is G4P. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence as set forth in SEQ ID NO:9, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence as set forth in SEQ ID NO:9 and contains a mutant G4P. In some embodiments, the PR3 antibody binding domain consists of the sequence as set forth in SEQ ID NO:9.

[0077] In some embodiments, the PR3 antibody binding domain is a mutated PR3 antibody binding domain containing a mutation at position 44 compared to mature human PR3, e.g., mature human PR3 set forth in SEQ ID NO: 1. In some embodiments, the mutation is H44A. In some embodiments, the PR3 antibody binding domain comprises an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence set forth in SEQ ID NO: 10 and contains the mutation H44A. In some embodiments, the PR3 antibody binding domain consists of the sequence shown in SEQ ID NO:10.

[0078] In some provided embodiments, the PR3 antibody binding domain is encoded by a sequence set forth in any one of SEQ ID NOs: 11-20, or a sequence exhibiting at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 11-20.

[0079] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 11, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 11. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 11.

[0080] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 12, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 12. In some embodiments, the encoded PR3 antibody binding domain contains the mutation S176A, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0081] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 13, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 13. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 13.

[0082] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 14, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 14. In some embodiments, the encoded PR3 antibody binding domain contains the mutation D91N, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0083] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 15, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 15.

[0084] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 16, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 16.

[0085] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 17. In some embodiments, the encoded PR3 antibody binding domain contains the mutation D175N, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0086] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 18. In some embodiments, the encoded PR3 antibody binding domain contains the mutation S176C, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0087] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO: 19. In some embodiments, the encoded PR3 antibody binding domain contains a mutation G4P, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0088] In some embodiments, the PR3 antibody binding domain is encoded by a nucleotide sequence set forth in SEQ ID NO:20, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:20. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the PR3 antibody binding domain is encoded by a sequence that consists of the sequence set forth in SEQ ID NO:20. In some embodiments, the encoded PR3 antibody binding domain contains the mutation H44A, based on the residue numbering of mature PR3 shown in SEQ ID NO:1.

[0089] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 1. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 11, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 11. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 11. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 11.

[0090] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO:2. In some such embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO:12, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO:12. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO:12. In some such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO:12.

[0091] In some such embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 3. In some such embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 13, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 13. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 13. In some such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 13.

[0092] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 4. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 14, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 14. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 14. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 14.

[0093] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 5. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 15, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 15. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 15. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 15.

[0094] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO:6. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO:16, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO:16. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO:16. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO:16.

[0095] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 7. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 17, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 17. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 17. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 17.

[0096] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 8. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 18, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 18. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 18.

[0097] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 9. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 19, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 19. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 19. In some embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 19.

[0098] In some embodiments, the PR3 antibody binding domain is or contains the sequence shown in SEQ ID NO: 10. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 20, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 20. In some embodiments, the PR3 antibody binding domain is or contains the amino acid sequence encoded by SEQ ID NO: 20. In some such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 20.

[0099] Exemplary PR3 antibody binding domains (or autoantibody binding domains) and corresponding SEQ ID NOs are listed in Table 2 below.

[0100] [Table 2]

[0101] B. Spacer In some embodiments, the CAAR disclosed herein further comprises a spacer (in some cases, also referred to as a spacer region). In some embodiments, the spacer region is located between the extracellular domain that comprises or consists of an antibody binding or autoantibody recognition component (e.g., PR3 antibody binding domain) and the transmembrane domain (also referred to herein as a transmembrane region).

[0102] In some embodiments, the length of the spacer is adjusted to optimize the biophysical synaptic distance between the CAAR-expressing cell and the target of the CAAR (e.g., an anti-PR3 antibody, e.g., a PR3 autoantibody). In some embodiments, the CAAR is expressed by a T cell, and the length of the spacer is adjusted to a length that is compatible with T cell activation or to optimize CAAR T cell performance.

[0103] In some embodiments, the spacer is selected such that the CAAR has less than 20% tonic signaling as assessed using the assay described in Example 2. In some embodiments, the tonic signaling is less than 15%, e.g., as assessed using the assay described in Example 2. In some embodiments, the tonic signaling is less than 10%, e.g., as assessed using the assay described in Example 2. In some embodiments, the spacer is selected such that the CAAR can kill at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the anti-PR3 antibody-expressing cell targets. In some embodiments, the CAAR can kill at least 65% of the anti-PR3 antibody-expressing cell targets as assessed using the assay described in Example 3.

[0104] In some embodiments, the spacer can be of a length that provides increased cellular responsiveness following antigen binding compared to the absence of the spacer or compared to an alternative spacer of a different length (e.g., longer in length).

[0105] Exemplary spacers include those having at least 10 or about 10-300 or about 300 amino acids, 10 or about 10-229 or about 229 amino acids, 10 or about 10-200 or about 200 amino acids, 10 or about 10-175 or about 175 amino acids, 10 or about 10-150 or about 150 amino acids, 10 or about 10-125 or about 125 amino acids, 10 or about 10-100 or about 100 amino acids, 10 or about 10-75 or about 75 amino acids, 10 or about 10-50 or about 50 amino acids, 10 or about 10-40 or about 40 amino acids, 10 or about 10-30 or about 30 amino acids, 10 or about 10-20 or about 20 amino acids, 10 or about 10-15 or about 15 amino acids, and any integer between the endpoints of any of the recited ranges. Exemplary spacers include those having at least or about 50-175 or about 175 amino acids, 50 or about 50-150 or about 150 amino acids, 10 or about 10-125 or about 125 amino acids, 50 or about 50-100 or about 100 amino acids, 100 or about 100-300 or about 300 amino acids, 100 or about 100-250 or about 250 amino acids, 125 or about 125-250 or about 250 amino acids, or 200 or about 200-250 or about 250 amino acids, and any integer between any endpoints of the recited ranges. In some embodiments, the spacer is at least or about 12 amino acids, at least 119 or about 119 amino acids, at least 125 or about 125 amino acids, at least 200 or about 200 amino acids, or at least 220 or about 220 amino acids, or at least 225 or about 225 amino acids in length. In some embodiments, the spacer is at least or about 13 amino acids in length, at least or about 120 amino acids, at least or about 120 amino acids, at least or about 125 amino acids, at least or about 200 amino acids, or at least or about 220 amino acids, or at least or about 229 amino acids in length.In some embodiments, the spacer is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids or less in length. In some embodiments, the spacer is at least 100 or about 100 amino acids in length, for example, at least 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 235, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 7 230, 240, or 250 amino acids, or about 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length.

[0106] In some embodiments, the spacer is at least 125 or about 125-300 or about 300 amino acids, 125 or about 125-250 or about 250 amino acids, 125 or about 125-230 or about 230 amino acids, 125 or about 125-200 or about 200 amino acids, 125 or about 125-180 or about 180 amino acids, 125 or about 125-150 or about 150 amino acids, 150 or about 150-300 or about 300 amino acids, 150 or about 150-250 or about 250 amino acids, 150 or about 150-230 or about 230 amino acids, 150 or about 150-200 or about 200 amino acids, 150 or about 1 The length is 50 to 180 or about 180 amino acids, 180 or about 180 to 300 or about 300 amino acids, 180 or about 180 to 250 or about 250 amino acids, 180 or about 180 to 230 or about 230 amino acids, 180 or about 180 to 200 or about 200 amino acids, 200 or about 200 to 300 or about 300 amino acids, 200 or about 200 to 250 or about 250 amino acids, 200 or about 200 to 230 or about 230 amino acids, 230 or about 230 to 300 or about 300 amino acids, 230 or about 230 to 250 or about 250 amino acids, or 250 or about 250 to 300 or about 300 amino acids. In some embodiments, the spacer is at least or about 129, 130 or about 130, 140 or about 140, 150 or about 150, 160 or about 160, 170 or about 170, 180 or about 180, 190 or about 190, 200 or about 200, 210 or about 210, 220 or about 220, 221 or about 221, 222 or about 222, 223 or about 223, 224 or about 224, 225 or about 225, 226 or about 226, 227 or about 227, 228 or about 228, 229 or about 229, or 230 or about 230 amino acids in length, or a length between any of the foregoing.

[0107] In some embodiments, the spacer may be or include at least a portion of an immunoglobulin constant region or a mutant or modified version thereof, such as a hinge region. In some embodiments, the constant region or portion is that of a human IgG, e.g., IgG4, IgG2, or IgG1. In some embodiments, the portion of an immunoglobulin constant region includes a hinge region (e.g., an IgG4 hinge region), as well as, where appropriate, a C H 1 / C L , C H 2 and / or C H 3 and / or one or more additional domains of a constant region, such as an Fc region. In some embodiments, a portion of the constant region serves as a spacer between the autoantibody recognition component, e.g., the PR3 antibody binding domain, and the transmembrane domain.

[0108] Exemplary spacers include an IgG hinge alone, H 2 and C H IgG hinge linked to one or more of the three domains, or C H In some embodiments, the spacer comprises an IgG hinge linked to the C3 domain. In some embodiments, the spacer comprises an IgG hinge alone. In some embodiments, the spacer comprises an IgG hinge, C H 2 and / or C H 3 can be derived from all or a portion of IgG4 or IgG2, such as all or a portion of human IgG4 or human IgG2. In some embodiments, the spacer comprises a hinge, C from IgG4, IgG2, and / or IgG2 and IgG4. H 2 and / or C H The hinge region may be a chimeric polypeptide containing one or more of the three sequences(s). In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region. In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region and / or an IgG2 hinge region, where the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; H 2 regions, IgG4 C H 2 domain and / or IgG2 CH IgG4 C H The two regions are suitably human IgG4 C H 2 domains, IgG2 C H The two regions are suitably human IgG2 C H 2 regions; and / or C H The three regions are IgG4 C H All or part of the three regions, and / or IgG2 C H Contains all or part of the three regions, IgG4 C H The three regions are suitably human IgG4 C H 3 regions, IgG2 C H The three regions are suitably selected from human IgG2 C H In some embodiments, the hinge, C H 2 and C H 3 is the hinge region from IgG4, C H 2 and C H In some embodiments, the hinge region is chimeric and comprises hinge regions derived from human IgG4 and human IgG2; H The two regions are chimeric and are derived from human IgG4 and human IgG2. H 2 regions; and / or C H The three regions are chimeric and are derived from human IgG4 and human IgG2. H In some embodiments, the spacer comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge that contains at least one amino acid substitution compared to the human IgG4 hinge region; H 2 domains; and human IgG4 C H Includes 3 areas.

[0109] In some embodiments, the spacer may contain one or more single amino acid mutations in one or more domains of the immunoglobulin, such as one or more domains of the hinge, CH2 or CH3 region. In some examples, the amino acid modification is a substitution of proline (P) with serine (S) in the hinge region of IgG4. In some embodiments, the spacer is or contains an IgG4 hinge, which is a mutant IgG4 hinge region containing a substitution of amino acids CPSC to CPPC compared to the wild-type IgG4 hinge region. In some embodiments, the spacer contains a CH2 of IgG4 that is a mutant CH2, and the amino acid modification is a substitution of C, ... H N177Q mutation at position 177 in the C2 region, or the full-length IgG2 Fc sequence shown in SEQ ID NO: 133, H and asparagine (N) to glutamine (Q) substitutions such as the N176Q mutation at position 176 in region 2.

[0110] In some examples, the spacer is 12 or about 12 amino acids in length, or no more than 12 or about 12 amino acids in length. In some examples, the spacer is 15 or about 15 amino acids in length, or no more than 15 or about 15 amino acids in length.

[0111] In some embodiments, the spacer comprises or consists of all or a portion of an immunoglobulin hinge, or a modified version thereof. In some embodiments, the spacer is 15 or about 15 amino acids or less in length. In some embodiments, the spacer comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof, and / or comprises about 15 or less amino acids. In some embodiments, the spacer is 13 or about 13 amino acids in length and / or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof. In some embodiments, the spacer is 12 or about 12 amino acids in length and / or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof. In some embodiments, the spacer is a nucleotide sequence of the formula X 1 PPX 2 P (SEQ ID NO: 124), 1 is glycine, cysteine ​​or arginine, and X 2 is cysteine ​​or threonine. In some embodiments, the spacer does not include the CD28 extracellular region or the CD8 extracellular region. In certain cases, the spacer has a methionine residue at the C-terminus. In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 22, 23, 125, 126, 127, 128, 129, 130, or 131, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO: 22. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO: 23.

[0112] In some embodiments, the spacer is, for example, a C of a human immunoglobulin, such as IgG4 and / or IgG2. Hor comprises an IgG hinge linked to 3 domains. In some embodiments, the spacer is 111 or about 111, 112 or about 112, 113 or about 113, 114 or about 114, 115 or about 115, 116 or about 116, 117 or about 117, 118 or about 118, 119 or about 119, 120 or about 120, 121 or about 121, 122 or about 122, 123 or about 123, 124 or about 124, or 125 or about 125 amino acids in length, or has a length between any of the foregoing. In some embodiments, the spacer is 119 or about 119 or 120 or about 120 amino acids in length. In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 24 or 96, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24 or 96. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO: 24. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO: 96. In some embodiments, the spacer is 120 or about 120 amino acids in length.

[0113] In some embodiments, the spacer may be derived in whole or in part from IgG4 and / or IgG2 and may contain mutations, such as one or more single amino acid mutations, in one or more domains. In some examples, the amino acid modification is a substitution of proline (P) with serine (S) in the hinge region of IgG4. In some embodiments, the spacer is or contains an IgG4 hinge, which is a mutant IgG4 hinge region that contains a substitution of amino acids CPSC to CPPC compared to the wild-type IgG4 hinge region. In some embodiments, the amino acid modification is a substitution of amino acid CPSC to CPPC in the C of the full-length IgG4 Fc sequence shown in SEQ ID NO: 132 to reduce glycosylation heterogeneity. H N177Q mutation at position 177 in the C2 region, or the full-length IgG2 Fc sequence shown in SEQ ID NO: 133, H and asparagine (N) to glutamine (Q) substitutions such as the N176Q mutation at position 176 in region 2.

[0114] In some embodiments, the spacer is an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric hinge H 2 region; and IgG4 C H or is a spacer as set forth in SEQ ID NO: 25 or 97. In some embodiments, the spacer is or contains an amino acid sequence having at least, or about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 25 or 97. In some embodiments, the spacer is or contains a sequence as set forth in SEQ ID NO: 25 or 97.

[0115] Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135, or International Publication No. WO2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity upon expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or reduce the likelihood of splice events at splice sites.

[0116] In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:23. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:97. In some embodiments, the spacer comprises or consists of the amino acid sequence set forth in SEQ ID NO:25.

[0117] In some embodiments, the spacer comprises or consists of an amino acid sequence exhibiting at least, or about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23, 96, or 97. In some such embodiments, the spacer is encoded by a polynucleotide optimized for codon usage and / or to reduce RNA heterogeneity, as appropriate. In some embodiments, the spacer comprises or consists of an amino acid sequence exhibiting at least, or about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22, 24, or 25. In some such embodiments, the spacer is encoded by a polynucleotide optimized for codon usage and / or to reduce RNA heterogeneity, as appropriate.

[0118] In some embodiments, the CAAR comprises a hinge domain derived from a CD8 polypeptide. In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 101, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 101. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO:101.

[0119] In some embodiments, the amino acid sequence of the spacer comprises or consists of (GGGGS)n. In some such embodiments, the spacer has 5-30 amino acids. In some embodiments, n is 1-6, inclusive. In one embodiment, n=1 (SEQ ID NO:99). In another embodiment, n=2. In another embodiment, n=3 (SEQ ID NO:100).

[0120] In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 99, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the spacer is or contains the sequence set forth in SEQ ID NO:99.

[0121] In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 100, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 100. In some embodiments, the spacer is or contains the sequence shown in SEQ ID NO: 100.

[0122] In some embodiments, the spacer is a flexible linker spacer. In some embodiments, the amino acid sequence of the spacer comprises or consists of EAAAK (SEQ ID NO: 102).

[0123] In some embodiments, the spacer comprises or consists of the sequence of SEQ ID NO: 102, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer is or contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity thereto. In some embodiments, the spacer is or contains the sequence shown in SEQ ID NO: 102.

[0124] C. Transmembrane domain In some embodiments, the extracellular binding domain containing the PR3 antibody binding component is linked to one or more intracellular signaling components (e.g., signaling components that mimic activation through a TCR complex) via a transmembrane domain (also referred to herein as a transmembrane region). Thus, in some embodiments, the PR3 antibody binding domain and spacer are linked to an intracellular signaling domain that includes one or more transmembrane domains, such as those described herein, and one or more intracellular components, such as those described herein. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the receptor is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0125] The transmembrane domain in some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane domain includes (i.e., includes at least the transmembrane domain(s)) derived from the alpha, beta or zeta chain of T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and / or CD154. For example, the transmembrane domain can be the CD28 transmembrane domain that includes the amino acid sequence shown in SEQ ID NO: 26 or 134. In some embodiments, the receptor transmembrane domain is the transmembrane domain of human CD28 or a variant thereof, such as the 27 amino acid transmembrane domain of human CD28 (Accession Number: P10747.1), or a 28 amino acid sequence, or a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 26 or 134, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 26 or 134. In some embodiments, the receptor's transmembrane domain is the transmembrane domain of human C8a or a variant thereof, such as the 21 amino acid transmembrane domain of human CD8a (Accession Number: P01732), or a sequence of 22 amino acids, or a transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 98 or 135, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 98 or 135.

[0126] In some embodiments, the transmembrane domain is encoded by a polynucleotide that is optimized for codon usage and / or to reduce RNA heterogeneity, for example by removing cryptic splice sites, as appropriate. In certain cases, the transmembrane domain has a methionine residue at the N-terminus.

[0127] In some embodiments, the transmembrane domain is or contains SEQ ID NO: 26 or 134, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent sequence identity to SEQ ID NO: 26 or 134. In some embodiments, the transmembrane domain is or contains the sequence set forth in SEQ ID NO: 26 or 134. In some embodiments, the transmembrane domain is or contains SEQ ID NO: 98 or 135, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent sequence identity to SEQ ID NO: 98 or 135. In some embodiments, the transmembrane domain is or contains the sequence set forth in SEQ ID NO: 98 or 135

[0128] Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises mainly hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer and / or transmembrane domain(s).

[0129] D. Intracellular signaling domains CAARs generally include an intracellular signaling domain that includes at least one intracellular signaling component(s). Some intracellular signaling domains include those that mimic or approximate signals through natural antigen receptors, signals through such receptors in combination with costimulatory receptors, and / or signals through costimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker, e.g., one that contains glycine and serine, e.g., a glycine-serine doublet, that is 2-10 amino acids in length, is present to form a link between the transmembrane domain and the intracellular signaling domain of the CAAR.

[0130] In some embodiments, the CAAR comprises an intracellular component or signaling domain of the TCR complex, such as the TCR CD3 chain, e.g., the CD3 zeta (CD3-ζ) chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, the PR3 antibody binding domain is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 intracellular signaling domain and / or other CD transmembrane domains. In some embodiments, the CAAR further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16.

[0131] In some embodiments, upon ligation of the CAAR, the cytoplasmic domain or intracellular signaling region of the CAR stimulates and / or activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the CAR. For example, in some situations, the CAAR induces a T cell function, e.g., cytolytic activity, such as secretion of cytokines or other factors, or T-helper activity. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or a costimulatory molecule is used instead of a complete immunostimulatory chain, e.g., when it translates an effector function signal. In some embodiments, the intracellular signaling domain(s) comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also includes that of a co-receptor that, in the natural context, acts in concert with such receptor to initiate signal transduction after antigen receptor binding, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same functional capability.

[0132] In the context of natural TCR, complete activation generally requires not only TCR-mediated signal transduction, but also costimulatory signal.Therefore, in some embodiments, CAAR also comprises components for generating secondary or costimulatory signal to promote complete activation.In other embodiments, CAAR does not comprise components for generating costimulatory signal.In some aspects, additional CAAR is expressed in the same cell and provides components for generating secondary or costimulatory signal.

[0133] It has been described that T cell activation is mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, CAAR comprises one or both of these classes of cytoplasmic signaling sequences.

[0134] In some aspects, the CAAR comprises a primary cytoplasmic signaling sequence that regulates the primary stimulation and / or activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that contain a primary cytoplasmic signaling sequence include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, the intracellular signaling region in the CAAR contains a cytoplasmic signaling domain, a part thereof, or a sequence derived from CD3 zeta. In some embodiments, the CD3 zeta comprises the sequence of amino acids shown in SEQ ID NO: 28, 136, or 137. In some embodiments, the CD3 zeta is encoded by a polynucleotide that is optimized for codon usage and / or to reduce RNA heterogeneity, for example, by removing cryptic splice sites as appropriate. In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain, such as the 112AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession Number: P20963.2), or a functional variant thereof, or a CD3 zeta signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling domain comprises a sequence of amino acids set forth in SEQ ID NO:28, 136, or 137, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:28, 136, or 137. In some embodiments, the CD3 zeta is or contains the sequence set forth in SEQ ID NO:28.

[0135] In some embodiments, the CAAR comprises a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and / or a transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule. Exemplary costimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS. For example, the costimulatory molecule may be derived from 4-1BB and may comprise the amino acid sequence shown in SEQ ID NO:27. In some embodiments, the 4-1BB-derived costimulatory molecule is encoded by a polynucleotide that is optimized for codon usage and / or to reduce RNA heterogeneity, for example, by removing cryptic splice sites. In some embodiments, the 4-1BB-derived costimulatory molecule comprises the amino acid sequence shown in SEQ ID NO:27. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as a 42 amino acid cytoplasmic domain of human 4-1BB (Accession Number: Q07011.1) or a functional variant or portion thereof, such as a sequence of amino acids set forth in SEQ ID NO:27, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:27. In some embodiments, the costimulatory molecule can be derived from CD28 and can comprise the amino acid sequence set forth in SEQ ID NO:138, encoded by the nucleotide sequence set forth in SEQ ID NO:139. In some embodiments, the intracellular signaling domain comprises an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a 41 amino acid domain thereof and / or such a domain having an LL to GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise a sequence of amino acids set forth in SEQ ID NO: 138 or 140, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 138 or 140.In some embodiments, the same CAR comprises both a stimulatory or activating component (e.g., a cytoplasmic signaling sequence) and a costimulatory component.

[0136] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, for example, any of those described herein. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0137] In some embodiments, the stimulatory or activating component is contained within one CAAR, while the costimulatory component is provided by another CAAR that recognizes another antigen. In some embodiments, the CAAR comprises an activating or stimulating CAAR and a costimulatory CAAR, both of which are expressed on the same cell (see International Publication No. WO2014 / 055668). In some aspects, the PR3-CAAR is a stimulatory or activating CAAR; in other aspects, it is a costimulatory CAAR. In some embodiments, the cell further comprises an inhibitory CAAR (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), whereby the stimulatory or activating signal delivered via the PR3-CAAR is reduced or inhibited by the binding of the inhibitory CAAR to its ligand, for example, reducing off-target effects.

[0138] In some embodiments, the two receptors induce activation and inhibitory signals to cells, respectively, so that the binding of one of the receptors to the antigen activates the cell or induces a response, but the binding of the second inhibitory receptor to the antigen induces a signal that suppresses or attenuates the response.An example is the combination of activating CAAR and inhibitory CAAR.Such a strategy can be used to reduce the possibility of off-target effects in the situation where, for example, activating CAAR binds to an antigen that is expressed in disease or condition but also expressed on normal cells, and inhibitory receptor binds to a separate antigen that is expressed on normal cells but not on disease or condition cells.

[0139] In some embodiments, the chimeric receptor comprises an intracellular component that is or comprises an inhibitory CAAR and attenuates or suppresses immune responses, such as ITAM and / or costimulatory promotion responses, in cells. Examples of such intracellular signaling components are those found on immune checkpoint molecules, such as PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2 / 4 adenosine receptor, including A2AR. In some embodiments, engineered cells comprise an inhibitory CAAR that comprises or is derived from the signaling domain of such inhibitory molecules, for example, to help attenuate cellular responses induced by activation and / or costimulatory CAAR.

[0140] In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and 4-1BB (CD137; TNFRSF9) costimulatory domain linked to a CD3 zeta intracellular domain.

[0141] In some embodiments, the CAR includes one or more, e.g., two or more, costimulatory domains and a stimulatory or activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3-zeta and 4-1BB.

[0142] E. Exemplary CAAR Provided herein is a CAAR that comprises any of PR3 antibody binding domains, such as any of those described herein, IgG4 hinge spacer, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:22; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:26; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:29, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:29. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:32 and contains the mutation S176A.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:35, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:35 and contains an unprocessed C-terminus. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:38, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:38 and contains the mutation D91N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:41 and contains an unprocessed N-terminus.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:44 and contains an N-terminal Ile deletion. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:47, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:47 and contains the mutation D175N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:50, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:50 and contains the mutation S176C.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:53, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:53 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:56 and contains the mutation H44A.

[0143] In some embodiments, the antibody binding domain of PR3 CAAR comprises a mutant PR3 antibody binding domain containing a mutation at position 91 compared to mature human PR3. In some embodiments, the mutation of the PR3 antibody binding domain is D91N. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 4, and a spacer having an amino acid sequence as shown in SEQ ID NO: 23. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 4, and a spacer having an amino acid sequence as shown in SEQ ID NO: 22. In some embodiments, the PR3 CAAR has an amino acid sequence as shown in SEQ ID NO: 38.

[0144] In some embodiments, the antibody binding domain of PR3 CAAR comprises a mutant PR3 antibody binding domain containing a mutation at position 44 compared to mature human PR3. In some embodiments, the mutation of the PR3 antibody binding domain is H44A. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 23. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 22. In some embodiments, the PR3 CAAR has an amino acid sequence as shown in SEQ ID NO: 56.

[0145] Provided herein is a CAAR that comprises any of PR3 antibody binding domains, such as any of those described herein, IgG4 hinge spacer, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:24; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:26; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:30, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:30. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:33, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:33 and contains the mutation S176A.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:36, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:36 and contains an unprocessed C-terminus. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:39, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:39 and contains the mutation D91N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:42, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:42 and contains an unprocessed N-terminus.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:45 and contains an N-terminal Ile deletion. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48 and contains the mutation D175N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:51, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:51 and contains the mutation S176C.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:54, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:54 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:57 and contains the mutation H44A.

[0146] In some embodiments, the antibody binding domain of PR3 CAAR comprises a mutant PR3 antibody binding domain containing a mutation at position 44 compared to mature human PR3. In some embodiments, the mutation of the PR3 antibody binding domain is H44A. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 96. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 24. In some embodiments, the PR3 CAAR has an amino acid sequence as shown in SEQ ID NO: 57.

[0147] Provided herein is a CAAR that comprises any of PR3 antibody binding domains, such as any of those described herein, IgG4 hinge spacer, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:25; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:26; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:31, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:34, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:34 and contains the mutation S176A.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:37, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:37 and contains an unprocessed C-terminus. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:40, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:40 and contains the mutation D91N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:43, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:43 and contains an unprocessed N-terminus.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:46 and contains an N-terminal Ile deletion. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:49, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:49 and contains the mutation D175N. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:52, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:52 and contains the mutation S176C.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:55 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:58 and contains the mutation H44A.

[0148] In some embodiments, the antibody binding domain of PR3 CAAR comprises a mutant PR3 antibody binding domain containing a mutation at position 4 compared to mature human PR3. In some embodiments, the mutation of the PR3 antibody binding domain is G4P. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO:9, and a spacer having an amino acid sequence as shown in SEQ ID NO:97. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO:9, and a spacer having an amino acid sequence as shown in SEQ ID NO:25. In some embodiments, the PR3 CAAR has an amino acid sequence as shown in SEQ ID NO:55.

[0149] In some embodiments, the antibody binding domain of PR3 CAAR comprises a mutant PR3 antibody binding domain containing a mutation at position 44 compared to mature human PR3. In some embodiments, the mutation of the PR3 antibody binding domain is H44A. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 97. In some embodiments, the PR3 CAAR comprises an antibody binding domain having an amino acid sequence as shown in SEQ ID NO: 10, and a spacer having an amino acid sequence as shown in SEQ ID NO: 25. In some embodiments, the PR3 CAAR has an amino acid sequence as shown in SEQ ID NO: 56.

[0150] Provided herein is a CAAR that comprises any of PR3 antibody binding domains, such as any of those described herein, IgG4 hinge spacer, CD8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:22; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:110, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:110 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:118, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:118 and contains the mutation H44A.

[0151] Provided herein is a CAAR that comprises any of the PR3 antibody binding domains, such as any of those described herein, an IgG4 hinge spacer, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.In certain embodiments, the CAAR comprises any of the PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:24; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 109, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 109 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:117, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:117 and contains the mutation H44A.

[0152] CAAR is provided herein, which comprises any of PR3 antibody binding domains, for example, any of those described herein, IgG4 hinge spacer, CD8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:101; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 107, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 107 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:115, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:115 and contains the mutation H44A.

[0153] Provided herein is a CAAR that comprises any of the PR3 antibody binding domains, such as any of those described herein, spacer, CD8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of the PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:102; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 108, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 108 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:116, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:116 and contains the mutation H44A.

[0154] Provided herein is a CAAR that comprises any of the PR3 antibody binding domains, such as any of those described herein, spacer, CD8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of the PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:100; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 103, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:111, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:111 and contains the mutation H44A.

[0155] Provided herein is a CAAR that comprises any of the PR3 antibody binding domains, such as any of those described herein, spacer, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of the PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:100; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:26; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 104, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 104 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:112, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:112 and contains the mutation H44A.

[0156] CAAR is provided herein, which comprises any of PR3 antibody binding domains, for example, any of those described herein, spacer, CD8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of PR3 antibody binding domains described herein; spacer that is or contains the sequence shown in SEQ ID NO:99; transmembrane domain that is or contains the sequence shown in SEQ ID NO:98; and intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 105, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 105 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:113, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:113 and contains the mutation H44A.

[0157] Provided herein is a CAAR that comprises any of the PR3 antibody binding domains, such as any of those described herein, spacer, CD28 transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.In certain embodiments, CAAR comprises any of the PR3 antibody binding domains described herein; a spacer that is or contains the sequence shown in SEQ ID NO:99; a transmembrane domain that is or contains the sequence shown in SEQ ID NO:26; and an intracellular signaling domain that comprises the 4-1BB signaling domain sequence shown in SEQ ID NO:27 and the CD3 zeta signaling domain sequence shown in SEQ ID NO:28. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106 and contains a mutant G4P. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:114, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:114 and contains the mutation H44A.

[0158] In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO:119, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:119 and contains the mutation H44A. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 120 and contains the mutation H44A. In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 121, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121 and contains the mutation H44A.In some of any of the embodiments, the PR3 CAAR comprises an amino acid sequence set forth in SEQ ID NO: 122, or an amino acid sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 122 and contains the mutation H44A.

[0159] In any of the provided embodiments, the PR3 CAAR is encoded by a sequence set forth in SEQ ID NOs:59-88 and 144-159, or a sequence exhibiting at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NOs:59-88 and 144-159. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:59, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:59. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:60, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:60.In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:61, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:61.

[0160] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:62, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:62. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:63, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:63. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:64, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:64.

[0161] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:65, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:65. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:66, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:66. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:67, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:67.

[0162] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:68, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:68. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:69, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:69. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:70, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:70.

[0163] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:71, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:71. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:72, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:72. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:73, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:73.

[0164] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:74, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:74. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:75, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:75. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:76, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:76.

[0165] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:77, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:77. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:78, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:78. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:79, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:79.

[0166] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:80, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:80. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:81, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:81. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:82, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:82.

[0167] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:83, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:83. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:84, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:84. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:85, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:85.

[0168] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:86, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:86. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:87, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:87. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:88, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:88.

[0169] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:144, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:144. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:145, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:145. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:146, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:146.

[0170] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:147, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:147. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:148, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:148. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:149, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:149.

[0171] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:150, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:150. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:151, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:151. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:152, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:152.

[0172] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:153, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:153. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:154, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:154. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:155, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:155.

[0173] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:156, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:156. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:157, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:157. In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:158, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:158.

[0174] In some of any of the embodiments, the PR3 CAAR is encoded by a nucleotide sequence set forth in SEQ ID NO:159, or a nucleotide sequence that is at least or about 85%, 86% or about 86%, 87% or about 87%, 88% or about 88%, 89% or about 89%, 90% or about 90%, 91% or about 91%, 92% or about 92%, 93% or about 93%, 94% or about 94%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:159.

[0175] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 29. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 59, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 59. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 59.

[0176] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 30. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 60, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 60. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 60.

[0177] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 31. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 61 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 61. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 61.

[0178] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 32. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 62 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 62 and containing the mutation S176A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 62.

[0179] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 33. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 63 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 63 and containing the mutation S176A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 63.

[0180] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 34. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 64, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 64 and containing the mutation S176A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 64.

[0181] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 35. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 65, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 65 and containing an unprocessed C-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 65.

[0182] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 36. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 66, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 66 and containing an unprocessed C-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 66.

[0183] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 37. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 67 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 67 and containing an unprocessed C-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 67.

[0184] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 38. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 68 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 68 and containing the mutation D91N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 68.

[0185] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 39. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 69, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 69 and containing the mutation D91N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 69.

[0186] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 40. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 70, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 70 and containing the mutation D91N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 70.

[0187] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 41. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 71 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 71 and containing an unprocessed N-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 71.

[0188] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 42. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 72 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 72 and containing an unprocessed N-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 72.

[0189] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 43. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 73 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 73 and containing an unprocessed N-terminus. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 73.

[0190] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 44. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 74, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 74 and containing an N-terminal Ile deletion. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 74.

[0191] In some of such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 45. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 75, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 75 and containing an N-terminal Ile deletion. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 75.

[0192] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 46. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 76, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 76 and containing an N-terminal Ile deletion. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 76.

[0193] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 47. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 77 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 77 and containing the mutation D175N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 77.

[0194] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 48. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 78 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 78 and containing the mutation D175N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 78.

[0195] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 49. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 79 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 79 and containing the mutation D175N. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 79.

[0196] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 50. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 80, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 80 and containing the mutation S176C. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 80.

[0197] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 51. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 81 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 81 and containing the mutation S176C. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 81.

[0198] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 52. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 82, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 82 and containing the mutation S176C. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 82.

[0199] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 53. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 83 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 83 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 83.

[0200] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 54. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 84 or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 84 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 84.

[0201] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 55. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 55 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 55 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 55 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 55 and contains a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO:85 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:85 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO:85.

[0202] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 56. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 86, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 86 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 86.

[0203] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 57. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 57 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 57 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 57 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 57 and contains the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO:87 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:87 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO:87.

[0204] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 58. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 88 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 88 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 88.

[0205] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 103. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 144, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 144 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 144.

[0206] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 104. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 104 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 104 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 104 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 104 and contains a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 145 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 145 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 145.

[0207] In some of such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 105. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 146, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 146 and containing a mutant G4P. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 146.

[0208] In some of such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 106. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 147, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 147 and containing a mutant G4P. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 147.

[0209] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 107. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 148, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 148 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 148.

[0210] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 108. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 149, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 149 and containing a mutant G4P. In some of such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 149.

[0211] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 109. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 150, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 150 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 150.

[0212] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 110. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 110 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 110 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 110 and contains a mutant G4P. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 110 and contains a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 151 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 151 and containing a mutant G4P. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 151.

[0213] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 111. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 and contains the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 152 or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 152 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains an amino acid sequence encoded by SEQ ID NO: 152.

[0214] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 112. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 153, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 153 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 153.

[0215] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 113. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 154, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 154 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 154.

[0216] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 114. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 155, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 155 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 155.

[0217] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 115. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 156, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 156 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 156.

[0218] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 116. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 157, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 157 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 157.

[0219] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 117. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 158, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 158 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 158.

[0220] In some of any such embodiments, the PR3 CAAR is or contains the sequence shown in SEQ ID NO: 118. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 159, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percent identity to SEQ ID NO: 159 and containing the mutation H44A. In some of any such embodiments, the PR3 CAAR is or contains the amino acid sequence encoded by SEQ ID NO: 159.

[0221] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 119. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 90% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 119 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 95% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 119 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 97% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 119 and contains the mutation H44A. In some embodiments, the PR3 CAAR comprises an amino acid sequence that is at least 99% or about this percentage sequence identity to the amino acid sequence set forth in SEQ ID NO: 119 and contains the mutation H44A.

[0222] In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 120. In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 121. In some of any such embodiments, the PR3 CAAR is or contains the sequence set forth in SEQ ID NO: 122.

[0223] In some embodiments, the CAAR-expressing cells provided exhibit biological activities or functions including cytotoxic activity, cytokine production, and the ability to proliferate.

[0224] In some embodiments, the biological or functional activity, e.g., cytotoxic activity, of the chimeric receptor can be measured using any of a number of known methods. Activity can be evaluated or determined either in vitro or in vivo. In some embodiments, activity can be evaluated once the cells are administered to a subject (e.g., a human). The parameters evaluated include specific binding of engineered or natural T cells or other immune cells to a target binding partner (e.g., PR3-ANCA), e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method, such as the cytotoxicity assay described in the Examples herein. In some embodiments, the provided CAAR can kill at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% of cellular targets expressing anti-PR3 antibodies. In some embodiments, the CAAR can kill at least 65% of cellular targets expressing anti-PR3 antibodies, as assessed using the assay described in Example 3. Other cytotoxicity assays are known in the art, such as those described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of cells may also be measured by assaying the expression and / or secretion of certain cytokines, such as interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or TGF-beta (TGFβ).Assays for measuring cytokines are well known in the art and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric bead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample. In some embodiments, biological activity is measured by assessing a clinical outcome.

[0225] In some embodiments, some of the CAARs provided exhibit target-dependent activity or signaling, i.e., signaling activity that is not measurably present or is at background level in the absence of target, e.g., PR3-ANCA.Thus, in some aspects, the CAARs provided do not exhibit tonic signaling, or exhibit only background or acceptable or low levels.Tonic signaling refers to CAAR activity (e.g., cytotoxic activity or other functional activity, such as cytokine production) that can occur with or without target, such as PR3-ANCA, and is target-independent activity or signaling.In some embodiments, among the multiple cells in the composition expressing CAAR, 10% or about 10%, 9% or about 9%, 8% or about 8%, 7% or about 7%, 5% or about 5%, 4% or about 4%, 3% or about 3%, 2% or about 2%, or less than 1% or about 1% of the multiple cells exhibit tonic signaling and / or antigen-independent activity or signaling. In some embodiments, antigen-independent activity or signaling is represented by off-target killing, as described in Example 8.

[0226] In some aspects, reporter cell lines can be employed to monitor antigen-independent activity and / or tonic signaling through CAAR-expressing cells. In some embodiments, T cell lines, such as Jurkat cell lines, contain reporter molecules, such as fluorescent proteins, such as red fluorescent protein, or other detectable molecules, expressed under the control of endogenous Nur77 transcriptional regulatory elements. In some embodiments, Nur77 reporter expression is cell-intrinsic and depends on signaling through recombinant reporters that contain primary activation signals in T cells, signaling domains of T cell receptor (TCR) components, and / or signaling domains that contain immunoreceptor tyrosine-based activation motifs (ITAMs), such as CD3ζ chain. Expression of Nur77 is generally not affected by other signaling pathways, such as cytokine signaling or Toll-like receptor (TLR) signaling, and may act cell-extrinsically and not depend on signaling through recombinant receptors. Thus, only cells expressing exogenous CAAR containing the appropriate signaling region can express Nur77 upon stimulation (e.g., binding of a specific antigen). In some cases, Nur77 expression may also exhibit a dose-dependent response to the amount of stimulus (e.g., antigen).

[0227] An exemplary reporter assay for evaluating tonic signaling is described in Example 2. In some embodiments, CAAR-transduced cells show antigen-specific activation after exposure to anti-human PR3 antibody. In some embodiments, anti-human PR3 antibody is MAB684022.

[0228] F. Polynucleotides Provided herein is a polynucleotide that encodes a therapeutic protein that contains the PR3 antibody binding domain provided herein. Also provided is a polynucleotide that encodes the CAAR described herein. Polynucleotides can include those that contain naturally occurring and / or non-naturally occurring nucleotides and bases, including those that have backbone modifications. The terms "nucleic acid molecule", "nucleic acid", "sequence of nucleotides" and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides can contain natural and / or non-natural nucleotides, including but not limited to DNA, RNA, and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide.

[0229] Provided is a polynucleotide that contains the nucleic acid that encodes any of the PR3 antibody binding domain constructs described herein.Also provided is a polynucleotide that contains the nucleic acid that encodes any of the PR3 CAARs described herein.In some embodiments, PR3 CAAR contains multiple domains, and all of the CAARs are encoded in more than one polynucleotide, such as two or more polynucleotides.In some embodiments, the polynucleotide is contained in a vector.

[0230] In some cases, the polynucleotide encoding PR3 CAAR contains a signal sequence encoding a signal peptide, in some cases encoded upstream of the nucleic acid sequence encoding PR3 CAAR or connected at the 5' end of the nucleic acid sequence encoding the antigen binding domain. In some cases, the polynucleotide containing the nucleic acid sequence encoding PR3 CAAR contains a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a natural polypeptide. In other aspects, the signal sequence may encode a heterologous or non-natural signal peptide. In some aspects, non-limiting exemplary signal peptides include the signal peptide of CD33 signal peptide shown in SEQ ID NO: 21. In some aspects, non-limiting exemplary signal peptides include the signal peptide of PR3 signal peptide shown in SEQ ID NO: 123. In some cases, the polynucleotide encoding PR3 CAAR can contain nucleic acid sequences encoding additional molecules such as surrogate or other markers, or can contain additional components such as promoters, regulatory elements, and / or multicistronic elements. In some embodiments, the nucleic acid sequence encoding PR3 CAAR can be operably linked to any of the additional components.

[0231] In some embodiments, the polynucleotide contains a nucleic acid sequence encoding any of the PR3 CAARs described in Section IF. In some embodiments, the nucleic acid sequence includes any of those described in Section IF.

[0232] In some embodiments, a polynucleotide is provided that contains a nucleic acid sequence encoding a PR3 antibody binding domain construct or a PR3 CAAR containing a PR3 antibody binding domain construct. In some of any of the embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain construct that includes a sequence set forth in any one of SEQ ID NOs: 11-20, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to any one of SEQ ID NOs: 11-20.

[0233] In some of any of the embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 11, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 12, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12. In some of any of the embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 13, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 14, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO:15, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:15.

[0234] In some of any of the embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 16, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 16. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 17, or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or about this percentage identity to SEQ ID NO: 17. In some of any of the embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 18, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO: 19, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19. In some embodiments, the polynucleotide contains a nucleic acid encoding a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO:20, or a nucleic acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:20.

[0235] Also provided are polynucleotides that are optimized for codon usage and / or to eliminate splice sites, such as cryptic splice sites. In some embodiments, the polynucleotides are modified to optimize codon usage. In some embodiments, the polynucleotides are codon optimized for expression in human cells, such as human T cells, e.g., primary human T cells. In some embodiments, the polynucleotides, e.g., those encoding any of the PR3 constructs or PR3 CAARs provided herein, are modified or modified (such as by optimization methods) to reduce heterogeneity or contain one or more nucleic acid sequences observed herein to provide improved characteristics of the polypeptide, such as CAAR, compared to those containing different reference sequences or those that are not optimized. In some embodiments, the polynucleotides are optimized by splice site elimination. Among such characteristics are improvements in RNA heterogeneity, such as those resulting from the presence of one or more splice sites, such as one or more cryptic splice sites, and / or improved expression and / or surface expression of the encoded protein, e.g., increased levels, uniformity, or consistency of expression among cells engineered to express the polypeptide or different therapeutic cell compositions. In some embodiments, the polynucleotides may be codon optimized for expression in human cells.

[0236] Genomic nucleic acid sequences are generally naturally processed in mammalian cells either simultaneously with or immediately after transcription, and the nascent precursor messenger ribonucleic acid (pre-mRNA) transcribed from genomic deoxyribonucleic acid (DNA) sequences is in some cases edited by splicing to remove introns and subsequently link exons in eukaryotic cells. Although consensus sequences of splice sites are known, in some embodiments, the specific nucleotide information defining splice sites may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on standard consensus sequences and are variably activated. Thus, variable splicing of pre-mRNA at cryptic splice sites results in heterogeneity of transcribed mRNA products upon expression in eukaryotic cells.

[0237] The polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences such as complementary DNA (cDNA) or parts thereof that do not contain introns. Therefore, splicing of such sequences is not expected to occur. However, the presence of hidden splice sites within the cDNA sequence may result in unintended or undesired splicing reactions and heterogeneity of transcribed mRNA. Such heterogeneity results in the translation of unintended protein products, such as truncated protein products with variable amino acid sequences that show modified expression and / or activity.

[0238] In some embodiments, the elimination of splice sites, for example, hidden splice sites, can improve or optimize the expression of transgene products, for example, polypeptides translated from transgenes, such as PR3 CAAR polypeptides. Splicing of the encoded transgene, such as the encoded PR3 CAAR molecule, at hidden splice sites can result in reduced protein expression, for example, reduced expression on the cell surface, and / or reduced function, for example, reduced intracellular signal transduction. In some embodiments, the polynucleotide encoding the PR3 CAAR protein is optimized to reduce or eliminate hidden splice sites. In some embodiments, the polynucleotide encoding the PR3 CAAR protein is optimized for codon expression, and / or has one or more sequences, such as those identified by the methods or observations herein regarding splice sites, and / or does not have an identified splice site, such as any of the splice sites identified herein.

[0239] RNA heterogeneity can be determined by any of the many methods provided or described herein or known. In some embodiments, the RNA heterogeneity of the transcribed nucleic acid is determined by amplifying the transcribed nucleic acid, such as by reverse transcriptase polymerase chain reaction (RT-PCR), and then detecting one or more differences, such as size differences, in one or more amplification products. In some embodiments, the RNA heterogeneity is determined based on the number of amplification products of different sizes, or the proportion of amplification products of various different sizes. In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is collected from a cell, expressing an optimized transgene, and amplified by reverse transcription polymerase chain reaction (RT-PCR) using a primer specific to the 5' untranslated region (5' UTR) located upstream of the transgene in the transcribed RNA, which in some cases corresponds to a part of the promoter sequence in the expression vector, and a primer specific to the 3' untranslated region (3' UTR) located downstream of the transgene expressed in the transcribed RNA sequence, or a primer specific to a sequence within the transgene. In certain embodiments, at least one primer complementary to a sequence in the 5' untranslated region (UTR) and at least one primer complementary to a sequence in the 3' untranslated region (UTR) are employed to amplify the transgene. RNA, such as messenger RNA, can be degraded and its heterogeneity analyzed by several methods. Non-limiting, exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field-flow fractionation, and chromatography, such as size-exclusion or liquid chromatography.

[0240] In some embodiments, the polynucleotide provided herein encoding the PR3 CAAR or PR3 construct provided herein comprises a modification to remove one or more splice donor and / or acceptor sites that may contribute to splice events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, the polynucleotide provided is modified in one or more polynucleotides in the spacer region to eliminate or reduce splice events.

[0241] Polynucleotides containing nucleic acid sequences encoding the exemplary PR3 CAAR and any of the exemplary CAARs described herein, either in whole or in part, fragment, or domain, are provided. Also provided herein are polynucleotides encoding PR3 CAARs. In some embodiments, the CAAR may be encoded by more than one different polynucleotide, for example, two or more polynucleotides. In some of any such embodiments, the two or more polynucleotides may each contain a nucleic acid encoding a portion, fragment, or domain of the CAAR. In some cases, the polynucleotide also includes a CD33 signal sequence or a PR3 signal sequence. Exemplary PR3 CAAR polynucleotide sequences are set forth in SEQ ID NOs: 59-88 and 144-159, which encode the amino acid sequences set forth in SEQ ID NOs: 29-58 and 103-118.

[0242] In some optional embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NOs: 59-88 and 144-159, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in SEQ ID NOs: 59-88 and 144-159. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:59 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:59. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:59. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:60 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:60. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:60. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:61 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:61. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:61.

[0243] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:68 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:68. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:68. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:69 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:69. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:69. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 70, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 70. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 70.

[0244] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:83 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:83. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:83. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:84 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:84. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:84. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:85 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:85. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:85.

[0245] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:86 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:86. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:86. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 87 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 87. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:87. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 88, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 88. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO:88.

[0246] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 144 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 144. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 144. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 145, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 145. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 145. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 146, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 146. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 146.

[0247] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 147, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 147. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 147. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 148 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 148. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 148. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 149 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 149. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 149.

[0248] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 150, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 150. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 150. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 151, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 151. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 151. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 152, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 152. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 152.

[0249] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 153, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 153. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 153. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 154 or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 154. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 154. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 155, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 155. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 155.

[0250] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 156, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 156. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 156. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 157, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 157. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 157. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 158, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 158. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 158.

[0251] In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 159, or a sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 159. In some of any of the embodiments, the polynucleotide comprises a sequence set forth in SEQ ID NO: 159.

[0252] In some of the embodiments, the polynucleotide comprises a nucleic acid encoding a sequence set forth in SEQ ID NO: 29-58 or 103-122, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 29-58 or 103-122. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:29, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:29. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:29. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 30, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 30. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 30. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:31, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:31. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:31.

[0253] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 38, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 38. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 38. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 39, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 39. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 39. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:40, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:40. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:40.

[0254] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:53, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:53. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:53. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:54, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:54. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:54. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:55, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:55. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:55.

[0255] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:56, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:56. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:56. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:57, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:57. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:57. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:58, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:58. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:58.

[0256] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 103, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 103. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 103. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 104, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 104. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 104. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 105, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 105. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 105. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 106, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 106. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 106.

[0257] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 107, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 107. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 107. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 108, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 108. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 108. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 109, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 109. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 109. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 110, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 110. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 110.

[0258] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:111, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:111. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:111. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 112, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 112. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 112. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 113, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 113. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 113. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 114, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 114. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 114.

[0259] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 115, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 115. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 115. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 116, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 116. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 116. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 117, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 117. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 117. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 118, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 118. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 118.

[0260] In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 119, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 119. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 119. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 120, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 120. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 120. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 121, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 121. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 121. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 122, or a sequence encoding a polypeptide sequence that exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 122. In some of any of the embodiments, the polynucleotide comprises a nucleic acid encoding the sequence set forth in SEQ ID NO: 122.

[0261] II. Engineered Cells Also provided are cells, such as engineered cells, that contain CAAR, for example, cells that contain the extracellular domain that comprises the PR3 antibody binding domain construct described herein.Also provided are populations of such cells, compositions that contain such cells, and / or compositions that are enriched for such cells, such as cells that express PR3 CAAR constitute at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more percent of the total cells in the composition, or a certain type of cells, such as T cells or CD8+ or CD4+ cells.Among the compositions are pharmaceutical compositions and formulations for administration, such as adoptive cell therapy.Also provided are therapeutic methods for administering cells and compositions to subjects, such as patients.

[0262] Also provided is a genetically engineered cell that expresses PR3 CAAR. The cell is generally a eukaryotic cell, such as a mammalian cell, typically a human cell. In some embodiments, the cell is a cell of the immune system, such as a cell of innate or adaptive immunity, such as a bone marrow cell or a lymphatic cell, derived from blood, bone marrow, lymph, or lymphoid organ, including, for example, lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cell is typically a primary cell, such as a cell that is directly isolated from a subject and / or that is isolated from a subject and frozen.

[0263] In certain embodiments, the cell expressing PR3 CAAR is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is an iPSC-derived cell differentiated from an iPSC.

[0264] In some embodiments, IPC differentiates into T cells or NK cells.In some embodiments, the method for differentiating iPSC into T cells or NK cells is described in, for example, published PCT applications International Publication No. WO2022120334, International Publication No. WO2022216514 and International Publication No. WO2022216624.In certain embodiments, iPSC can be differentiated by any method known in the art. Exemplary methods are described in US Patent No. 8846395, US Patent No. 8945922, US Patent No. 8318491, International Publication No. WO2010 / 099539, International Publication No. W02012 / 109208, International Publication No. W02017 / 070333, International Publication No. WO2017 / 179720, International Publication No. W02016 / 010148, International Publication No. WO2018 / 048828 and International Publication No. WO2019 / 157597, each of which is incorporated herein by reference in its entirety. In some embodiments, the differentiated T cells are gamma-delta T cells. In some embodiments, the differentiated T cells are alpha-beta T cells. The differentiation protocol may use feeder cells or may be feeder-free. As used herein, "feeder cells" or "feeders" are terms describing one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cells may grow, expand, or differentiate, as the feeder cells provide stimuli, growth factors, and nutrients for the support of the second cell type.

[0265] In some embodiments, the iPSC-derived cells are NK cells prepared by a method for differentiating iPSC cells into NK cells by subjecting the cells to a differentiation protocol that includes the addition of recombinant human IL-12p70 for the final 24 hours of culture.

[0266] In some embodiments, the iPSCs are modified by genomic engineering. The use of iPSCs allows cell engineering to generate controlled cell banks of modified cells that can be expanded and differentiated into desired immune effector cells to provide large quantities of homogenous allogeneic therapeutic products. In some embodiments, the genomic engineering includes targeted editing, which may include, but is not limited to, deletions, insertions, or in / dels performed by CRISPR, ZFN, TALEN, homing nucleases, homologous recombination, or any other functional variation of these methods. In some embodiments, the genomic engineering includes one or more exogenous polynucleotides that are integrated into one or more loci on the chromosomes of the iPSCs. In some embodiments, the iPSCs or cells differentiated therefrom are engineered with genomic modifications that enhance the therapeutic properties of the derived cells. In some embodiments, the resulting differentiated cells derived from the engineered iPSCs contain modifications that render the cells hypoimmune for use as an allogeneic cell product. Exemplary genomic modifications are described, for example, in published PCT applications WO2022120334, WO2022216514 and WO2022216624.

[0267] In some embodiments, iPSCs or iPSC-derived cells (e.g., T cells or NK cells) are modified by one or more gene edits (e.g., knockout) or insertion (e.g., knockin) of one or more exogenous genes to render the cells hypoimmune. In some embodiments, the cells are modified by deletion of one or more target genes or insertion of one or more exogenous genes to render the cells hypoimmune or to modulate their activity.

[0268] In some embodiments, the cells are allogeneic cells, hi some embodiments, the cells are engineered to be in a hypoimmune state.

[0269] In some embodiments, the cells are autologous cells.

[0270] In some embodiments, the cells are T cells. In some embodiments, the cells are selected from the total T cell population, CD4 T cells, CD4+ ... + cells, CD8 + The present invention includes one or more subsets of T cells or other cell types, such as cells, and subpopulations thereof. In some embodiments, provided herein is a T cell comprising a CAAR. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell.

[0271] The cells can be allogeneic and / or autologous with respect to the subject to be treated. Among these methods are off-the-shelf methods. In some aspects, such as off-the-shelf techniques, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from a subject, preparing, processing, culturing, and / or manipulating them, and reintroducing them into the same patient before or after cryopreservation, as described herein.

[0272] T cells and / or CD4 + and / or CD8 + T cell subtypes and subpopulations include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (T SCM ), central memory T(T CM ), Effector Memory T(T EM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0273] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0274] Also provided herein is a cell population comprising CAAR, from which the cells of the population are provided. In some embodiments, the cell population is a cell derived from the peripheral blood of a subject, such as a subject to be treated (e.g., by autologous adoptive cell therapy). In some embodiments, the cell population is obtained by apheresis. In some embodiments, the cell population is obtained by leukapheresis. In some embodiments, the cell population is a peripheral blood mononuclear cell (PBMC). In some embodiments, among the cells in the population, 30% or more than about 30%, 40% or more than about 40%, 50% or more than about 50%, 60% or more than about 60%, 70% or more than about 70% of the cells are T cells.

[0275] Also provided herein is a T cell population comprising a CAAR from which the T cells of the population are provided. In some embodiments, the T cell population is enriched, such as by selection, from a biological sample (e.g., blood, leukapheresis or apheresis sample) from a subject. In some embodiments, the T cell population is enriched for CD3+ cells. In some embodiments, the T cell population contains more than or about 75% CD3+ T cells, such as more than or about 80% CD3+ T cells, more than or about 85% CD3+ T cells, more than or about 90% CD3+ T cells, or more than or about 95% CD3+ T cells. In some embodiments, the T cell population contains more than or about 95% CD3+ T cells. In some embodiments, the T cell population contains CD4+ and CD8+ T cells. In some embodiments, the CD4+ and CD8+ T cells are present at a ratio of 1:3 or about 1:3 to 3:1, e.g., 1:2 to 2:1, e.g., 1:1 or about 1:1. In some embodiments, the T cell population contains more than or about 75% CD4+ and CD8+ T cells, e.g., more than or about 80% CD4+ and CD8+ T cells, more than or about 85% CD4+ and CD8+ T cells, more than or about 90% CD4+ and CD8+ T cells, or more than or about 95% CD4+ and CD8+ T cells. In some embodiments, the T cell population contains more than or about 95% CD4+ and CD8+ T cells.

[0276] In some embodiments, at least 25% of the cells in the population express CAAR.In some embodiments, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 50% or about 50%, at least 60% or about 60%, at least 70% or about 70%, at least 80% or about 80%, or at least 90% or about 90% or more of the cells in the population express CAAR.In some embodiments, expression refers to the positive surface expression of CAAR on the cell surface, for example, as determined by flow cytometry.

[0277] In some embodiments, the cell contains one or more polynucleotides introduced through genetic engineering, thereby expressing recombinant or genetically engineered products of such polynucleotides. In some embodiments, the polynucleotide is heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as, for example, from another organism or cell that is not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the polynucleotide is not naturally occurring, and is a polynucleotide not found in nature, including, for example, those that contain a chimeric combination of polynucleotides encoding various domains from multiple different cell types. In some embodiments, the cell (e.g., engineered cell) contains a vector (e.g., a viral vector, an expression vector, etc.) described herein, such as a vector that contains a nucleic acid encoding the PR3 CAAR described herein.

[0278] A. Vectors and Methods for Genetic Engineering Also provided are methods, nucleic acids, compositions and kits for expressing PR3 CAAR and for generating genetically engineered cells that express said CAAR.In some embodiments, one or more CAARs can be genetically engineered into cells or multiple cells.Genetic engineering generally involves the introduction of nucleic acids that code for recombinant or engineered components into cells, for example, by retroviral transduction, transfection or transformation.

[0279] Nucleic acids may include those that include naturally occurring and / or non-naturally occurring nucleotides and bases, including those with backbone modifications. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides that comprises a nucleic acid molecule or polynucleotide. In some aspects, the nucleic acid sequence encoding at least a portion of an antibody or antigen-binding fragment thereof, such as an scFv, conjugate, receptor (e.g., CAAR), provided herein, can be optimized, e.g., codon-optimized for expression in human cells, and / or optimized to reduce or eliminate cryptic splice sites.

[0280] In some embodiments, the polynucleotide also comprises one or more additional sequences, such as those encoding a marker, or one or more additional molecules, such as a promoter, a regulatory element, and / or a multicistronic element. In some embodiments, the provided polynucleotide comprises any of the polynucleotides described herein.

[0281] Also provided is a nucleic acid, for example, a vector containing polynucleotide, and an engineered cell containing vector, for example, an engineered immune cell expressing PR3 CAAR.Also provided is a method for engineering cells, such as immune cells, to express PR3 CAAR.Nucleic acid can code for the amino acid sequence comprising wild-type PR3 antibody binding domain, or mutant PR3 antibody binding domain as extracellular antigen binding domain; transmembrane domain and intracellular domain, for example, CD3 zeta and costimulatory signaling domain.

[0282] In further embodiments, one or more vectors (e.g., expression vectors) comprising such polynucleotides are provided. In further embodiments, host cells comprising such polynucleotides are provided. In one such embodiment, the host cell comprises (e.g., is transformed with) a vector comprising a nucleic acid encoding an amino acid sequence comprising a PR3 CAAR. In some embodiments, one or more such host cells are provided. In some embodiments, compositions comprising one or more such host cells are provided. In some embodiments, one or more host cells can express different or the same PR3 CAAR. In some embodiments, each of the host cells can express more than one PR3 CAAR.

[0283] Also provided is a method for making PR3 CAAR.For recombinant production of CAAR, the nucleic acid sequence encoding CAAR, for example, as described herein, can be isolated and inserted into one or more vectors for further cloning and / or expression in host cell.Such nucleic acid sequence can be easily isolated and sequenced using conventional procedures.In some embodiments, a method for making PR3 CAAR is provided, which comprises culturing a host cell comprising the nucleic acid sequence encoding the antibody provided above under suitable conditions for expression of the receptor.

[0284] The provided embodiments further include vectors and host cells and other expression systems for expressing and producing the PR3 CAAR, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells, such as human cells, and cell-free expression systems.

[0285] In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining them with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of cytokines or activation markers, followed by transduction of the activated cells and expansion in culture to sufficient numbers for clinical application.

[0286] In some situations, overexpression of stimulatory factors (e.g., lymphokines or cytokines) can be toxic to the subject. Thus, in some situations, engineered cells contain gene segments that make the cells susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example, in some embodiments, cells are engineered to be eliminated as a result of changes in the in vivo conditions of the patient to whom they are administered. A negative selectable phenotype can result from the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound. Negative selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 2:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0287] In some embodiments, cells are further engineered to promote the expression of cytokines or other factors.Various methods for the introduction of genetically engineered components, such as PR3 CAAR, are well known and can be used with the provided methods and compositions.Exemplary methods include the method for the transfer of receptor-encoding polynucleotide, including by viral, such as retroviral or lentiviral transduction, transposon, and electroporation.

[0288] In some embodiments, recombinant polynucleotides are introduced into cells using vectors derived from recombinant infectious viral particles, such as Simian Virus 40 (SV40), adenovirus, or adeno-associated virus. In some embodiments, the recombinant polynucleotide is transferred into the T cells using a recombinant lentiviral vector, e.g., an HIV-1 lentiviral-based vector (lentivector; see, e.g., Amado et al., Science. 1999 Jul 30;285(5428):674-676); or a retroviral vector, e.g., a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

[0289] In some embodiments, the retroviral or lentiviral vector has a long terminal repeat (LTR). In some embodiments, the vector is derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), murine stem cell virus (MSCV), splenic focus forming virus (SFFV), human immunodeficiency virus type 1 (HIV-1) or human immunodeficiency virus type 2 (HIV-2 / SIV). In some embodiments, the vector is self-inactivating (SIN). In some embodiments, the vector is a conditionally replicating (mobilizable) vector. Most lentiviral vectors are derived from human, feline or simian lentiviruses. Most retroviral vectors are derived from murine retroviruses. In some embodiments, lentiviruses or retroviruses include those derived from any avian or mammalian cell source. Lentiviruses or retroviruses are typically amphotropic, meaning that they can infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and / or env sequences. Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701, Cooper et al. (2003) Blood. 101:1637-1644, Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114, and Cavalieri et al. (2003) Blood. 102(2): 497-505.A number of illustrative retroviral systems have also been described (see, e.g., Amado et al., (1999) Science 285(5428):674-676; U.S. Pat. Nos. 5,219,740, 6,207,453, and 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0290] In some embodiments, the recombinant polynucleotide is transferred to the T cell via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298, and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, the recombinant polynucleotide is transferred to the T cell via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437, Sharma et al. (2013) Molec Ther Nucl Acids 2, e74, and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston (1990) Nature 346: 776-777); and strontium phosphate DNA co-precipitation (Brash et al., (1987) Mol. Cell Biol.7: 2031-2034). Other approaches and vectors for the transfer of polynucleotides encoding recombinant products are described, for example, in International Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0291] Among the additional polynucleotides, e.g., genes for introduction, are genes that improve the outcome of the treatment, e.g., by promoting the survival and / or function of the transferred cells; genes that provide genetic markers for the selection and / or evaluation of cells, e.g., to evaluate in vivo survival or localization; genes that improve safety by making cells susceptible to negative selection in vivo, e.g., as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991), and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived from the fusion of dominant positive and negative selectable markers. See, e.g., Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0292] In some embodiments, the vector or construct may contain a promoter and / or enhancer or regulatory element for regulating the expression of the encoded recombinant receptor. In some examples, the promoter and / or enhancer or regulatory element may be a condition-dependent promoter, enhancer, and / or regulatory element. In some examples, these elements drive the expression of the transgene. In some examples, the CAR transgene may be operably linked to a promoter such as the EF1 alpha promoter with the HTLV1 enhancer (SEQ ID NO: 164). In some examples, the CAR transgene is operably linked to a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE; SEQ ID NO: 165) located downstream of the transgene.

[0293] In some embodiments, a vector or construct may contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, may be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, a transcription unit may be engineered as a bicistronic unit that contains an IRES (internal ribosome entry site), which allows for the simultaneous expression of gene products (e.g., encoding the first and second chimeric receptors) by messages from a single promoter. For example, in some embodiments, a vector or construct may contain a nucleic acid encoding an anti-ROR1 receptor (e.g., an anti-ROR1 CAR) provided herein and a nucleic acid encoding a different molecule, separated by an IRES, under the control of a single promoter.

[0294] Alternatively, in some cases, a single promoter can direct the expression of RNA containing two or three genes (e.g., encoding a first and a second binding molecule, e.g., an antibody recombinant receptor) in a single open reading frame (ORF) separated from each other by sequences encoding a self-cleaving peptide (e.g., a 2A cleavage sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide and is cleaved into individual proteins during (in the case of T2A) or after translation. In some cases, a peptide such as T2A can cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A, e.g., SEQ ID NO: 21 or 168), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20 or 167), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6, 17 or 166), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or 19), as described in U.S. Patent Publication No. 20070116690. In some embodiments, one or more distinct or separate promoters drive expression of one or more nucleic acid molecules encoding one or more binding molecules, e.g., recombinant receptors.

[0295] Any of the PR3 CAARs provided herein can be encoded by polynucleotides that contain one or more nucleic acid molecules that code for receptors in any combination or arrangement.For example, one, two, three or more polynucleotides can code for one, two, three or more different receptors or domains.In some embodiments, one vector or construct contains one or more nucleic acid molecules that code for PR3 CAARs, and a separate vector or construct contains the nucleic acid molecule that codes for additional PR3 CAARs.

[0296] In some embodiments, the nucleic acid molecule can also encode one or more surrogate marker(s), such as a fluorescent protein (e.g., green fluorescent protein (GFP)) or a cell surface marker (e.g., a truncated surface marker such as truncated EGFR (tEGFR)), which can be used to confirm the transduction or engineering of cells to express the receptor. For example, in some aspects, exogenous marker genes are utilized in conjunction with engineered cell therapy to allow for cell detection or selection, and in some cases also to facilitate cell suicide by ADCC. Exemplary marker genes include truncated epidermal growth factor receptor (EGFRt), which can be co-expressed with a transgene of interest (e.g., CAAR or TCR) in transduced cells (see, e.g., U.S. Pat. No. 8,802,374). EGFRt contains an epitope recognized by the antibody cetuximab (Erbitux®). For this reason, Erbitux® can be used to identify or select cells engineered with EGFRt constructs, including cells that have also been co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR).

[0297] In some embodiments, the nucleic acid encoding the binding molecule further contains a nucleic acid sequence encoding one or more marker(s). In some embodiments, the one or more marker(s) is a transduction marker, a surrogate marker and / or a selection marker.

[0298] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells into which a polynucleotide, for example, a polynucleotide encoding PR3 CAAR, has been introduced. In some embodiments, the transduction marker can indicate or confirm the modification of a cell. In some embodiments, the surrogate marker is a protein that is made to be co-expressed on the cell surface with PR3 CAAR. In certain embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes PR3 CAAR. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, for example, T2A, P2A, E2A, or F2A. Exogenous marker genes can be utilized in conjunction with engineered cells, in some cases to allow for cell detection or selection, and in some cases to promote cell suicide.

[0299] Exemplary surrogate markers may include truncated forms of cell surface polypeptides, e.g., truncated forms that are non-functional and do not or cannot transduce and / or internalize signals or signals normally transduced by the full-length form of the cell surface polypeptide. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences shown in SEQ ID NOs: 141-143) or prostate specific membrane antigen (PSMA) or modified forms thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which may be used to identify or select cells engineered with the tEGFR construct and the encoded exogenous protein, and / or to exclude or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434. In some embodiments, the marker, e.g., surrogate marker, comprises CD34, NGFR, CD19 or a truncated CD19, e.g., a truncated non-human CD19, or all or a portion (e.g., a truncated form) of an epidermal growth factor receptor (e.g., tEGFR).

[0300] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as superfold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), as well as variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or mutants thereof.

[0301] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to an exogenous substance or drug. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified versions thereof.

[0302] In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in PCT International Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include a sequence of amino acids set forth in SEQ ID NOs: 141-143, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 141-143.

[0303] In some embodiments, the marker is a molecule that is not naturally found on T cells or that is not naturally found on the surface of T cells, e.g., a cell surface protein, or portion thereof.

[0304] In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, ie, one that is not recognized as "self" by the immune system of the host into which the cells are adoptively transferred.

[0305] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than being used as a marker for genetic engineering, e.g., to select successfully engineered cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, e.g., a ligand for cells encountered in vivo, such as a co-stimulatory or immune checkpoint molecule to enhance and / or attenuate the response of cells upon adoptive transfer and encounter with the ligand.

[0306] Also provided are compositions containing one or more nucleic acid molecules, vectors or constructs, such as any of the above. In some embodiments, the nucleic acid molecules, vectors, constructs or compositions can be used to engineer cells, such as T cells, to express binding molecules, such as antibodies or recombinant receptors, and / or additional binding molecules.

[0307] B. Preparation of Cells for Manipulation In some embodiments, the preparation of engineered cells comprises one or more culture and / or preparation steps.The cells for CAAR introduction can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject.In some embodiments, the subject from which the cells are isolated is a subject that has a disease or condition, or requires or is administered cell therapy.The subject in some embodiments is a human being that requires a particular therapeutic intervention, such as adoptive cell therapy, from which the cells are isolated, processed, and / or engineered.

[0308] Thus, the cells in some embodiments are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from subjects, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with viral vectors), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or samples that are processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0309] In some embodiments, the sample from which the cells are derived or isolated is blood or blood-derived sample, or is or is derived from apheresis or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0310] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, e.g., mouse, rat, non-human primate, or pig.

[0311] In some embodiments, the isolation of cells includes one or more preparative and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, and lyse or remove cells that are sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity, and / or resistance to a particular component.

[0312] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample, in some embodiments, contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some embodiments, contains cells other than red blood cells and platelets.

[0313] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is accomplished by a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing, e.g., Ca++ / Mg++-free PBS. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in medium.

[0314] In some embodiments, the method comprises a density-based cell separation method, for example, preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0315] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation can be used. In some embodiments, the separation is affinity or immunoaffinity-based separation. For example, the isolation in some aspects comprises the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, for example by incubation with an antibody or binding partner that specifically binds to such marker, followed generally by a washing step and separation of the cells that are bound to the antibody or binding partner from the cells that are not bound to the antibody or binding partner.

[0316] Such separation step can be based on positive selection, in which the cells that bind to the reagent are retained for further use, and / or negative selection, in which the cells that do not bind to the antibody or binding partner are retained. In some cases, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, so that separation is best performed based on markers expressed by cells other than the desired population.

[0317] Separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker.Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0318] In some cases, multiple rounds of separation steps are performed, and the positively or negatively selected fraction from one step is subjected to another separation step, e.g., subsequent positive or negative selection. In some cases, a single separation step can deplete cells that express multiple markers simultaneously, e.g., by incubating cells with multiple antibodies or binding partners, each antibody or binding partner being specific for the marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0319] For example, in some embodiments, specific subpopulations of T cells, such as cells that express positive or high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques.

[0320] For example, CD3+,CD28+ T cells can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, MACSiBeads™, etc.).

[0321] In some embodiments, isolation is achieved by enrichment of a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by enriching for a particular cell population by positive selection, or depleting a particular cell population by negative selection, respectively. 高 ) This is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0322] In some embodiments, T cells are separated from the PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high extent on one or more naive, memory, and / or effector T cell subpopulations.

[0323] In some embodiments, the CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM ) Cell enrichment is undertaken to enhance certain characteristics, such as to improve long-term survival, expansion, and / or engraftment following administration, and in some aspects is particularly robust in such subpopulations (see Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701). In some embodiments, a combination of TCM-enriched CD8+ and CD4+ T cells further enhances the response.

[0324] In embodiments, memory T cells are present in both the CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0325] In some embodiments, the central memory T(T CM Enrichment for central memory T (T) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some embodiments, it is based on negative selection for cells expressing or high expressing CD45RA and / or granzyme B. In some embodiments, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one embodiment, central memory T (T CM) Enrichment of cells is performed starting from the negative fraction of cells selected on the basis of CD4 expression, which are subjected to negative selection on the basis of CD14 and CD45RA expression, and positive selection on the basis of CD62L. Such selections are performed simultaneously in some embodiments, and sequentially in either order in other embodiments. In some embodiments, the same selection step on the basis of CD4 expression used in preparing the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, as appropriate, after one or more additional positive or negative selection steps.

[0326] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, and both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on expre...

Claims

1. (a) an extracellular proteinase 3 (PR3) antibody-binding domain, wherein the extracellular PR3 antibody-binding domain is a mutant PR3 protein having reduced enzymatic activity compared to a wild-type PR3 protein having the sequence set forth in SEQ ID NO: 1; (b) a transmembrane region; and (c) Intracellular signal transduction region A chimeric autoantibody receptor (CAAR) comprising:

2. 2. The CAAR of claim 1, further comprising a spacer between the extracellular PR3 antibody binding domain and the transmembrane domain. Claim 3: The mutant PR3 protein comprises an amino acid substitution, wherein the amino acid substitution is selected from the group consisting of G4P, H44A, D91N, D175N, S176A, and S176C; or 2. The CAAR of claim 1, wherein the amino acid substitution is a conservative substitution of an amino acid substitution selected from the group consisting of G4P, H44A, D91N, D175N, S176A, and S176C.

4. A CAAR as described in claim 1, wherein the PR3 antibody binding domain comprises an amino acid sequence that exhibits at least 90% sequence identity with sequence number 9.

5. A CAAR as described in claim 1, wherein the PR3 antibody binding domain comprises an amino acid sequence that exhibits at least 90% sequence identity with sequence number 10.

6. A CAAR as described in claim 1, wherein the transmembrane region is or includes a transmembrane domain derived from CD4, CD28, or CD8.

7. The CAAR described in claim 1, wherein the intracellular signaling region comprises an intracellular signaling domain capable of inducing a primary activation signal in T cells.

8. A CAAR as described in claim 7, wherein the intracellular signaling domain is the cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain.

9. A CAAR as described in claim 7, wherein the intracellular signaling region further includes a costimulatory signaling region.

10. The CAAR described in claim 9, wherein the costimulatory signaling region includes an intracellular signaling domain of CD28, 4-1BB, or ICOS.

11. The spacer is (i) a hinge region of an immunoglobulin or a variant thereof; or (ii) the amino acid GGGGS (SEQ ID NO: 99) 3. The CAAR of claim 2, comprising:

12. The spacer is (i) an IgG4 hinge region or a variant thereof; (ii) a mutant IgG4 hinge region comprising a substitution of amino acids CPSC to CPPC compared to the wild-type IgG4 hinge region; (iii) an immunoglobulin hinge region and an immunoglobulin CH3 region; or (iv) an immunoglobulin hinge region, an immunoglobulin CH2 region or a chimeric CH2 region of two different immunoglobulins, and an immunoglobulin CH3 region; 12. The CAAR of claim 11, comprising:

13. The CAAR described in claim 1, wherein the CAAR comprises an amino acid sequence that exhibits at least 85% sequence identity with any of the sequences set forth in SEQ ID NOs: 29, 30, 31, 38, 39, 40, 53, 54, 55, 56, 57, 58, 104, 110, or 111.

14. (a) a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO:9; (b) a spacer comprising the sequence set forth in SEQ ID NO: 100; (c) a transmembrane region comprising the sequence set forth in SEQ ID NO: 26; (d) a costimulatory signaling region comprising the sequence set forth in SEQ ID NO: 27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO: 28 CAAR including.

15. A CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 104, wherein the sequence of amino acids comprises a G4P mutation, based on the numbering of positions in SEQ ID NO:

1.

16. (a) a PR3 antibody binding domain comprising the sequence set forth in SEQ ID NO:9; (b) a spacer comprising the sequence set forth in SEQ ID NO: 22; (c) a transmembrane region comprising the sequence set forth in SEQ ID NO: 98; (d) a costimulatory signaling region comprising the sequence set forth in SEQ ID NO: 27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO: 28 CAAR including.

17. A CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 110, wherein the sequence of amino acids comprises a G4P mutation, based on the numbering of positions in SEQ ID NO:

1.

18. (a) a PR3 antibody-binding domain comprising the sequence set forth in SEQ ID NO: 10; (b) a spacer comprising the sequence set forth in SEQ ID NO: 100; (c) a transmembrane region comprising the sequence set forth in SEQ ID NO: 98; (d) a costimulatory signaling region comprising the sequence set forth in SEQ ID NO: 27; and (e) an intracellular signaling region comprising the sequence shown in SEQ ID NO: 28 CAAR including.

19. A CAAR comprising a sequence of amino acids that exhibits at least 90% sequence identity with SEQ ID NO: 111 or 119, wherein the sequence of amino acids comprises an H44A mutation, based on the numbering of positions in SEQ ID NO:

1.

20. The CAAR can be specifically bound by an anti-PR3 anti-neutrophil cytoplasmic antibody (PR3-ANCA); and / or The CAAR of any one of claims 1 and 14 to 19, wherein the CAAR is specific for PR3-reactive B cells.

21. A polynucleotide comprising a nucleic acid encoding the CAAR of any one of claims 1 and 14 to 19.

22. A vector comprising the polynucleotide of claim 21.

23. A cell comprising the CAAR of any one of claims 1 and 14 to 19, a polynucleotide comprising a nucleic acid encoding the CAAR of any one of claims 1 and 14 to 19, or a vector comprising the polynucleotide.

24. The cell of claim 23, which is a lymphocyte.

25. A pharmaceutical composition comprising the cells of claim 23 for treating a disease or disorder in a subject, wherein the disease or disorder is an autoimmune condition.

26. 24. A method for killing PR3-reactive B cells, comprising contacting PR3-reactive B cells with a cell of claim 23 or a composition comprising a cell of claim 23, wherein the method is performed in vitro or ex vivo.

27. 24. Use of a cell according to claim 23 or a composition comprising a cell according to claim 23 in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the disease or disorder is an autoimmune condition.

28. 26. The pharmaceutical composition of claim 25, wherein the disease or disorder is selected from the group consisting of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), or eosinophilic granulomatosis with polyangiitis (EGPA).

29. The pharmaceutical composition of claim 25, wherein the disease or disorder is PR3-ANCA vasculitis and the subject has undergone remission induction therapy or a preconditioning therapy shown to be effective in depleting ANCA.

30. The pharmaceutical composition of claim 29, wherein the induction therapy includes cyclophosphamide.