Chimeric antigen receptor modified t-cells (car-t) for treatment of hematological and solid tumor cancers

Abstract

To provide chimeric antigen receptors that target human ROR-1, cell compositions expressing the chimeric antigen receptors, and methods and uses of the chimeric antigen receptors and / or the cell compositions.SOLUTION: Provided herein is a chimeric antigen receptor (CAR) and CAR-expressing immune cells that target human ROR1 expressed aberrantly on a tumor cancers. The CAR can be expressed by T lymphocytes isolated from an individual afflicted with cancer and readministered to the individual.SELECTED DRAWING: Figure 13

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 824,080, filed Mar. 26, 2019; U.S. Provisional Application No. 62 / 931,103, filed Nov. 5, 2019; and U.S. Provisional Application No. 62 / 969,569, filed Feb. 3, 2020, which are hereby incorporated by reference in their entirety and for all purposes.

[0002] Sequence Listing The sequence listing described in File 048537 - 623001WO_SEQUENCE_LISTING_ST25.TXT (created on Mar. 23, 2020, 69,632 bytes, machine format IBM - PC, MS - Windows operating system) is hereby incorporated by reference in this specification.

Background Art

[0003] The utility of chimeric antigen receptor-modified T cells (CAR-T) for treating cancer has been demonstrated in numerous clinical studies, including the FDA approval of two of these products for the treatment of pediatric acute lymphoblastic leukemia and transformed non-Hodgkin lymphoma. These successful CAR-Ts, tisagenlecleucel and axicabtagene ciloleucel, developed by Novartis and Kite Pharmaceuticals, respectively, are autologous products that target the B cell antigen CD-19. Although they target the same cancer antigen, the CAR-T products they created have different structures and manufacturing methods. In addition to these approved products, many additional CAR-T clinical trials, backed by various pharmaceutical and biotechnology companies at home and abroad, are underway, targeting the same antigen and involving unique structures and manufacturing methods similar to the approved products. In addition to CARs targeting CD-19, numerous additional cancer-related antigens are being targeted in ongoing clinical studies by CARs that employ diverse structures, T cell targets, activation and manufacturing methods, as well as patient pretreatment protocols. The numerous clinical studies employing these different agents and approaches have highlighted the complex pharmacology of CAR-T. The compositions and methods provided herein address the current needs in the art regarding ROR-1-specific CARs and their use in therapy and diagnosis. SUMMARY OF THE INVENTION

[0004] Disclosed herein are chimeric antigen receptors that target human ROR-1, cell compositions that express the chimeric antigen receptors, and methods and uses of the chimeric antigen receptors and / or cell compositions. The chimeric antigen receptors described herein can be isolated from an individual suffering from cancer and expressed by T lymphocytes that are re-administered to the individual. Administration of T cells that express the CARs described herein serves as an effective therapeutic treatment for cancers that express ROR-1. Further disclosed herein are optimized intracellular signaling domains and membrane spacers that result in improved function of anti-ROR-1 CAR-T cells.

[0005] Aspects disclosed herein provide chimeric antigen receptors that target ROR-1. The chimeric receptor comprises i) an antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1 and the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain; ii) a spacer domain, wherein the spacer domain comprises a spacer having a length of 10 to 240 amino acids; iii) a transmembrane domain; and iv) an intracellular domain. In some embodiments, the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48. Alternatively, the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54.

[0006] In some cases, the light chain variable domain is attached to the N-terminus or C-terminus of the heavy chain variable domain. In some embodiments, the light chain variable domain is covalently attached to the heavy chain variable domain via a polypeptide linker. In some embodiments, the polypeptide linker comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the light chain variable domain consists of the sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the heavy chain variable domain consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the light chain variable domain consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, the light chain variable domain consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 25. In some embodiments, the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 26.

[0007] In some cases, the spacer domain includes an antibody domain. In some embodiments, the antibody domain includes an immunoglobulin hinge domain, an immunoglobulin constant heavy chain 3 (CH3) domain, an immunoglobulin constant heavy chain 2 (CH2) domain, or a combination thereof. In some embodiments, the antibody domain consists of an immunoglobulin hinge domain. In some embodiments, the antibody domain consists of an immunoglobulin hinge domain and an immunoglobulin constant heavy chain 3 (CH3) domain. In some embodiments, the antibody domain consists of an immunoglobulin hinge domain, an immunoglobulin constant heavy chain 3 (CH3) domain, and an immunoglobulin constant heavy chain 2 (CH2) domain.

[0008] In some cases, the spacer domain is 14 to 120 amino acids in length. In some embodiments, the spacer domain includes the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42. In some embodiments, the spacer domain consists of the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0009] In some cases, the transmembrane domain includes a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain includes the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD28 transmembrane domain consists of the amino acid sequence of SEQ ID NO: 32.

[0010] In some cases, the intracellular domain includes an intracellular co-stimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof. In some embodiments, the intracellular co-stimulatory signaling domain is a 4-1BB intracellular co-stimulatory signaling domain, a CD28 intracellular co-stimulatory signaling domain, an ICOS intracellular co-stimulatory signaling domain, an OX-40 intracellular co-stimulatory signaling domain, or any combination thereof. In some embodiments, the 4-1BB intracellular co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the 4-1BB intracellular co-stimulatory signaling domain consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the intracellular co-stimulatory signaling domain comprises a CD28 intracellular co-stimulatory signaling domain and a 4-1BB intracellular co-stimulatory signaling domain. In some embodiments, the intracellular co-stimulatory signaling domain comprises an intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain. In some embodiments, the CD3ζ intracellular T cell signaling domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CD3ζ intracellular T cell signaling domain consists of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the chimeric antigen receptor binds to cells expressing ROR-1.

[0011] Aspects disclosed herein provide nucleic acids encoding the chimeric antigen receptors described herein. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0012] Aspects disclosed herein provide cells comprising the nucleic acids described herein. Aspects disclosed herein also provide cells that express the chimeric antigen receptors described herein. In some embodiments, the cells are T lymphocytes. In some embodiments, the T lymphocytes are CD4+ T lymphocytes or CD8+ T lymphocytes. In some embodiments, the cells are natural killer cells, genetically engineered natural killer cells, or CD56+ cells.

[0013] Aspects disclosed herein provide a pharmaceutical composition comprising a therapeutically effective amount of the cells described herein and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition is formulated for intravenous injection.

[0014] Aspects disclosed herein provide a method of treating cancer in an individual in need thereof by administering to the individual the pharmaceutical composition described herein. In some embodiments, the cancer comprises leukemia, lymphoma, chronic lymphocytic leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer. In some embodiments, the method further comprises administering siltuximab to the individual. In some embodiments, siltuximab and the pharmaceutical composition are administered separately.

[0015] Aspects disclosed herein provide a method of treating cancer in an individual in need thereof, or the use of a chimeric antigen receptor or chimeric antigen receptor T cell for a method of treating an individual cancer, wherein the cancer is a CD19-negative cancer or has reduced CD19 expression as a result of prior treatment targeting CD19. In some embodiments, the individual has been previously treated with a therapeutic agent targeting CD19. In some embodiments, the therapeutic agent targeting CD19 is an antibody that binds CD19. In some embodiments, the therapeutic agent targeting CD19 is a chimeric antigen receptor T cell that targets CD19. In some embodiments, the therapeutic agent targeting CD19 is a chimeric antigen receptor NK cell that targets CD19. In some embodiments, the cancer expresses ROR1.

[0016] Aspects disclosed herein provide a pharmaceutical composition described herein for use in a method of treating an individual's cancer. In some embodiments, the cancer includes leukemia, lymphoma, chronic lymphocytic leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] Definition Although various embodiments and aspects of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Those skilled in the art will be able to come up with many variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments described herein can be employed in the practice of the present invention.

[0019] The headings of the items used in this document are for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents (including but not limited to patents, patent applications, treatises, books, manuals, papers) cited in this application are hereby expressly incorporated by reference in their entirety for any purpose.

[0020] The abbreviations used in this specification have their conventional meanings within the chemical and biological arts. The chemical structures and formulas described herein are constructed in accordance with the standard rules of chemical valence known in the chemical art.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example, Singleton et al., Dictionary Of Microbiology And Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0022] As used herein, the term "about" means a range of values including the particular value and would be considered by one of ordinary skill in the art to be reasonably similar to the particular value. In embodiments, the term "about" means within one standard deviation of the measurement value generally accepted in the art. In embodiments, about means a range extending from ±10% of the particular value. In embodiments, about means the specified value.

[0023] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and their complements. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Nucleic acids as used herein also refer to nucleic acids having the same basic chemical structure as naturally occurring nucleic acids. Such analogs have modified sugars and / or modified peptide backbones, but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid mimics refer to compounds having a structure different from the general chemical structure of nucleic acids, but functioning in a manner similar to naturally occurring nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNA).

[0024] The term "gene" means a segment of DNA involved in the production of a protein, which includes regions before and after the coding region (leader and trailer), and intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements necessary during the transcription and translation of the gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0025] As used herein in reference to a gene, the terms "expression" or "expressed" mean the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on either the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by that DNA produced by the cell. The expression level of a non-coding nucleic acid molecule can be detected by standard PCR or Northern blot methods well known in the art. See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0026] Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression", the transfected gene is not passed on to daughter cells during cell division. Since the expression of the gene is limited to the transfected cells, the expression of the gene is lost over time. In contrast, co-transfecting the transfected cell with another gene that confers a convenience during selection and the transfected gene can result in stable expression of the transfected gene. The advantage of such selection can be resistance to a specific toxin presented to the cell.

[0027] The terms "transfection," "transduction," "transfect," or "transduce" can be used interchangeably throughout and are defined as the process of introducing nucleic acid molecules and / or proteins into cells. Nucleic acids can be introduced into cells using non-viral or virus-based methods. The nucleic acid molecule can be a gene sequence encoding a complete protein or a functional portion thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection through heat shock, magnetofection, and electroporation. In some embodiments, the nucleic acid molecule is introduced into cells using electroporation according to standard procedures well known in the art. In the case of virus-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, the nucleic acid molecule is introduced into cells using a retroviral vector according to standard procedures well known in the art. The terms "transfection" or "transduction" also refer to the introduction of proteins into cells from the external environment. Typically, protein transduction or transfection relies on the attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0028] The term "plasmid" or "expression vector" refers to a nucleic acid molecule encoding a gene and / or regulatory elements necessary for the expression of the gene. Expression of a gene from a plasmid can occur either cis or trans. When a gene is expressed cis, the gene and regulatory elements are encoded by the same plasmid. Trans expression refers to the case where the gene and regulatory elements are encoded by separate plasmids.

[0029] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are modified post-translationally, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a compound having a hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0030] Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be referred to by their generally accepted one-letter codes.

[0031] When used in the context of numbering of a given amino acid or polynucleotide sequence, the terms "numbered relative to" or "corresponding to" refer to the numbering of residues of a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. Amino acid residues of a protein "correspond to" a given residue if they occupy the same essential structural position within the same protein as the given residue. One of ordinary skill in the art will readily recognize the identity and position of residues corresponding to a particular position of a protein (e.g., ROR-1) in other proteins using different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., ROR-1), the identity and position of residues corresponding to a particular position of the protein are identified in other protein sequences that align with this protein. For example, a selected residue of a selected protein corresponds to glutamic acid at position 138 if the selected residue occupies the same essential spatial or other structural relationship to glutamic acid at position 138. In some embodiments, when the selected protein is aligned for maximum homology with the protein, the position in the aligned selected protein that aligns with glutamic acid 138 is said to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three-dimensional structure alignment can also be used, for example, where the structure of the selected protein is aligned for maximum correspondence with glutamic acid at position 138 and the overall structures are compared. In this case, the amino acid that occupies the same essential position as glutamic acid 138 in the structural model corresponds to the glutamic acid 138 residue.

[0032] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, and the polymer can optionally be conjugated to moieties not composed of amino acids. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "Fusion protein" refers to a chimeric protein that encodes two or more separate protein sequences expressed recombinantly as a single moiety.

[0033] The term "recombinant", when used with reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by, or is the result of, laboratory methods. Thus, for example, recombinant proteins include those produced by laboratory methods. A recombinant protein can contain amino acid residues not found within the native (non-recombinant) form of the protein, or can contain modified (e.g., labeled) amino acid residues.

[0034] The term "isolated", when applied to a nucleic acid or protein, means that the nucleic acid or protein is substantially free of other cellular components that are associated with it in its natural state. This can be, for example, in a homogeneous state and can be either dry or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is present as the major species in a preparation is substantially purified.

[0035] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that, when compared and aligned to maximize correspondence over a comparison window or specified region, using one of the following sequence comparison algorithms or by manual alignment and visual inspection, are identical or have amino acid residues or nucleotides that are identical at a particular percentage (i.e., for a particular region, such as the entire polypeptide sequence of the invention or an individual domain of the polypeptide of the invention, about 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity). Such sequences are said to be "substantially identical". This definition also refers to the complement of the test sequence. Optionally, the identity exists over a region of at least about 50 nucleotides, and more preferably over a region of 100 to 500 or 1000 or more nucleotides.

[0036] "Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide sequence or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences (excluding additions or deletions). This percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences, generating the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percent sequence identity.

[0037] In array comparison, typically, one array functions as a reference array and the test array is compared. When using an array comparison algorithm, the test and reference arrays are input into a computer, subarray coordinates are specified as needed, and the parameters of the array algorithm program are specified. Default program parameters can be used, or alternative parameters can be specified. The array comparison algorithm then calculates the percent sequence identity of the test array compared to the reference array based on the program parameters.

[0038] As used herein, a "comparison window" refers to any one segment of contiguous positions selected from the group consisting of, for example, the full-length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, and after the two sequences are optimally aligned, the sequences can be compared to a reference sequence of the same number of contiguous positions. Any method of aligning sequences for comparison well known in the art is contemplated. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0039] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated using parameters M (reward score for a pair of matching residues, always > 0) and N (penalty score for a mismatch residue, always < 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops from its maximum achieved value by an amount X, when the cumulative score goes to zero or below due to the accumulation of one or more residue alignments having negative scores, or when the end of either sequence is reached. The W, T, and X values of the BLAST algorithm parameters determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands.For amino acid sequences, the BLASTP program uses a word length of 3 and an expectation value (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses an alignment (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and a comparison of both strands as defaults.

[0040] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences could occur by chance. For example, if the smallest sum probability in a comparison of a test nucleic acid and a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0041] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody to the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically or substantially identical to a second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0042] Antibodies are large and complex molecules (molecular weight approximately 150,000, or approximately 1320 amino acids) with a complex internal structure. Natural antibody molecules contain a pair of two identical polypeptide chains, each pair having one light chain and one heavy chain. The light and heavy chains are each composed of two regions: a variable ("V") region involved in binding to the target antigen and a constant ("C") region that interacts with other components of the immune system. The variable regions of the light and heavy chains come together in three-dimensional space to form the variable region that binds to an antigen (e.g., a receptor on the surface of a cell). Within each variable region of the light or heavy chain, there are three short segments (average length of 10 amino acids) called complementarity-determining regions ("CDRs"). The six CDRs (three from the light chain and three from the heavy chain) of the antibody variable domain are folded together in three-dimensional space to form the actual antibody binding site and dock to the target antigen. The positions and lengths of the CDRs are precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The portions of the variable region that are not included in the CDRs are called the framework ("FR") and form the surroundings of the CDRs.

[0043] As used herein, an "antibody variant" refers to a polypeptide that can bind to an antigen and contains one or more structural domains of an antibody or a fragment thereof (e.g., a light chain variable domain, a heavy chain variable domain). Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fabs 2 , bispecific Fabs 2 , trispecific Fabs 3, a monovalent IgG, scFv, bispecific antibody, bispecific diabody, trispecific tribody, scFv-Fc, minibody, IgNAR, V-NAR, hcIgG, VhH, or peptibody. As used herein, "peptibody" refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by chondrichthyans or camels. General descriptions of antibodies and their variable regions from camels, as well as methods for their production, isolation, and use, can be found in International Publication No. WO97 / 49805 and International Publication No. WO97 / 49805, which are hereby incorporated by reference in their entirety for all purposes. Similarly, antibodies and their variable regions from chondrichthyans, as well as methods for their production, isolation, and use, can be found in International Publication No. WO2005 / 118629, which is hereby incorporated by reference in its entirety for all purposes.

[0044] As used herein, the terms "CDR L1", "CDR L2", and "CDR L3" refer to complementarity determining regions (CDRs) 1, 2, and 3 of the variable light chain (L) of an antibody. In some embodiments, the variable light chain presented herein includes CDR L1, CDR L2, and CDR L3 in the N-terminal to C-terminal direction. Similarly, as used herein, the terms "CDR H1", "CDR H2", and "CDR H3" refer to complementarity determining regions (CDRs) 1, 2, and 3 of the variable heavy chain (H) of an antibody. In some embodiments, the variable light chain presented herein includes CDR L1, CDR L2, and CDR L3 in the N-terminal to C-terminal direction.

[0045] The term "antibody" is used according to its generally known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as many well-characterized fragments produced by digestion with various proteases. Thus, for example, pepsin digests the antibody below the disulfide bonds in the hinge region to produce a dimer of Fab, which is a light chain bound to V H -C H1 and F(ab)' 2 . F(ab)' 2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab)' 2 dimer to Fab' monomers. Fab' monomers are essentially Fab with a part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Although various antibody fragments have been defined with respect to the digestion of intact antibodies, one of ordinary skill in the art will understand that such fragments can be synthesized de novo using chemical or recombinant DNA methodologies. Thus, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fv), or those identified using phage display libraries (e.g., see McCafferty et al., Nature 348:552-554 (1990)).

[0046] The term "antigen" provided herein refers to a molecule that can bind to the antibody-binding domain provided herein. The "antigen-binding domain" provided herein is the region of the antibody that binds to an antigen (epitope). As described above, the antigen-binding domain generally consists of one constant domain and one variable domain of each of the heavy and light chains (V L , V H , C L and C HIt consists of (1). The paratope or antigen-binding site is formed at the N-terminus of the antigen-binding domain. The two variable domains of the antigen-binding domain usually bind to an epitope on the antigen.

[0047] Antibodies exist, for example, as intact immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests the antibody below the disulfide bond in the hinge region to produce a dimer of Fab, which is a light chain bound to V H -C H1 and F(ab)' 2 2. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer to Fab' monomers. The Fab' monomer is an antigen-binding portion that essentially has a part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Although various antibody fragments have been defined for the digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo using chemical or recombinant DNA methodologies. Thus, as used herein, the term antibody also includes antibody fragments produced by modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fv), or those identified using phage display libraries (e.g., see McCafferty et al., Nature 348:552-554 (1990)).

[0048] A single-chain variable fragment (scFv) is usually a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, connected by a short linker peptide of 10 to about 25 amino acids. The linker usually contains a lot of glycine for flexibility and can contain a lot of serine or threonine for solubility. The linker connects the N-terminus of V H to the V LIt can be connected to the C-terminus thereof, or vice versa.

[0049] The epitope of an antibody is the region of its antigen to which the antibody binds. Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to its antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 30%, preferably 50%, 75%, 90%, or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody.

[0050] As used herein, the term "ROR-1" or "ROR1" refers to a recombinant or naturally occurring form of tyrosine kinase-like orphan receptor 1 (ROR-1) or a variant or homolog thereof that maintains ROR-1 activity (e.g., within a range of activity that is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to ROR-1). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 amino acid contiguous portion) compared to the naturally occurring ROR-1 protein. In some embodiments, the ROR-1 protein is substantially identical to the protein identified by accession number NP_005003.1 or a variant or homolog having substantial identity thereto. In embodiments, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 55. In embodiments, the ROR-1 protein is the amino acid sequence of SEQ ID NO: 55. In embodiments, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 56. In embodiments, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 57.

[0051] The terms "siltuximab", "UC-961", and "99961.1" refer to a humanized monoclonal antibody that can bind to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR-1). In embodiments, siltuximab is any one of the antibodies or fragments thereof disclosed in U.S. Patent Application No. 14 / 422,519, which is incorporated herein by reference in its entirety for all purposes.

[0052] As used herein, the term "CD28 transmembrane domain" refers to either a transmembrane domain of CD28 or a recombinant or naturally occurring form of a variant or homolog thereof that maintains CD28 transmembrane activity (e.g., within a range of activity of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CD28 transmembrane domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200 - amino acid contiguous portion) compared to the naturally occurring CD28 transmembrane domain peptide. In embodiments, the CD28 transmembrane domain is a human CD28 transmembrane domain protein. In embodiments, a variant or mutant of the CD28 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD28 transmembrane domain protein. In embodiments, a variant or mutant of the CD28 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD28 transmembrane domain protein. In embodiments, a variant or mutant of the CD28 transmembrane domain protein contains no deletions compared to the naturally occurring CD28 transmembrane domain protein. In embodiments, a variant or mutant of the CD28 transmembrane domain protein contains no insertions compared to the naturally occurring CD28 transmembrane domain protein. In embodiments, a variant or mutant of the CD28 transmembrane domain protein contains substitutions that are conservative substitutions compared to the naturally occurring CD28 transmembrane domain protein. In embodiments, the CD28 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001230006.1, or an isoform or naturally occurring variant or mutant thereof. In embodiments, the CD28 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001230007.1, or an isoform or naturally occurring variant or mutant thereof.In an embodiment, the CD28 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_006130.1, or an isoform or naturally occurring mutant or variant thereof.

[0053] As used herein, the term "CD4 transmembrane domain" refers to either a transmembrane domain of CD4 or a recombinant or naturally occurring form of a variant or homolog thereof that maintains CD4 transmembrane domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD4 transmembrane domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200 - amino acid contiguous portion) compared to the naturally occurring CD4 transmembrane domain polypeptide. In embodiments, the CD4 transmembrane domain is a human CD4 transmembrane domain protein. In embodiments, a variant or mutant of the CD4 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD4 transmembrane domain protein. In embodiments, a variant or mutant of the CD4 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD4 transmembrane domain protein. In embodiments, a variant or mutant of the CD4 transmembrane domain protein contains no deletions compared to the naturally occurring CD4 transmembrane domain protein. In embodiments, a variant or mutant of the CD4 transmembrane domain protein contains no insertions compared to the naturally occurring CD4 transmembrane domain protein. In embodiments, a variant or mutant of the CD4 transmembrane domain protein contains substitutions that are conservative substitutions compared to the naturally occurring CD4 transmembrane domain protein. In embodiments, the CD4 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_000607.1, or an isoform or naturally occurring variant or mutant thereof. In embodiments, the CD4 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001181943.1, or an isoform or naturally occurring variant or mutant thereof.In an embodiment, the CD4 transmembrane domain comprises all or part of a protein identified by NCBI sequence reference NP_001181944.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD4 transmembrane domain comprises all or part of a protein identified by NCBI sequence reference NP_001181945.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD4 transmembrane domain comprises all or part of a protein identified by NCBI sequence reference NP_001181946.1, or an isoform or naturally occurring variant or variant thereof.

[0054] As used herein, the term "CD8 transmembrane domain" refers to either the transmembrane domain of CD8 or a recombinant or naturally occurring form of a variant or homolog thereof that maintains CD8 transmembrane domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD8 transmembrane domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200 - amino acid contiguous portion) compared to the naturally occurring CD8 transmembrane domain polypeptide. In embodiments, the CD8 transmembrane domain is the CD8A transmembrane domain. In embodiments, the CD8 transmembrane domain is the CD8B transmembrane domain. In embodiments, the CD8 transmembrane domain is a human CD8 transmembrane domain protein. In embodiments, a variant or mutant of the CD8 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD8 transmembrane domain protein. In embodiments, a variant or mutant of the CD8 transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD8 transmembrane domain protein. In embodiments, a variant or mutant of the CD8 transmembrane domain protein contains no deletions compared to the naturally occurring CD8 transmembrane domain protein. In embodiments, a variant or mutant of the CD8 transmembrane domain protein contains no insertions compared to the naturally occurring CD8 transmembrane domain protein. In embodiments, a variant or mutant of the CD8 transmembrane domain protein contains substitutions that are conservative substitutions compared to the naturally occurring CD8 transmembrane domain protein. In embodiments, the CD8 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001139345.1, or an isoform or naturally occurring variant or mutant thereof.In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_001181943.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_001181944.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_001181945.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_741969.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_001759.3, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference XP_011531466.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_001171571.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_757362.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein specified by NCBI sequence reference NP_742100.1, or an isoform or naturally occurring variant or variant thereof.In an embodiment, the CD8 transmembrane domain comprises all or part of a protein identified by NCBI sequence reference NP_742099.1, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or part of a protein identified by NCBI sequence reference NP_004922.1, or an isoform or naturally occurring variant or variant thereof.

[0055] As used herein, the term "CD3-zeta transmembrane domain" refers to either the transmembrane domain of CD3-zeta or a recombinant or naturally occurring form of a variant or homolog thereof that maintains CD3-zeta transmembrane domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD3-zeta transmembrane domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200-amino acid contiguous portion) compared to the naturally occurring CD3-zeta transmembrane domain peptide. In embodiments, the CD3-zeta transmembrane domain is a human CD3-zeta transmembrane domain protein. In embodiments, a variant or mutant of the CD3-zeta transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD3-zeta transmembrane domain protein. In embodiments, a variant or mutant of the CD3-zeta transmembrane domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD3-zeta transmembrane domain protein. In embodiments, a variant or mutant of the CD3-zeta transmembrane domain protein contains no deletions compared to the naturally occurring CD3-zeta transmembrane domain protein. In embodiments, a variant or mutant of the CD3-zeta transmembrane domain protein contains no insertions compared to the naturally occurring CD3-zeta transmembrane domain protein. In embodiments, a variant or mutant of the CD3-zeta transmembrane domain protein contains substitutions that are conservative substitutions compared to the naturally occurring CD3-zeta transmembrane domain protein. In embodiments, the CD3-zeta transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_000725.1, or an isoform or naturally occurring variant or mutant thereof.In an embodiment, the CD3-zeta transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_932170.1, or an isoform or naturally occurring mutant or variant thereof.

[0056] As used herein, the term "CD28 co-stimulatory domain" refers to either a recombinant or naturally occurring form of the co-stimulatory domain of CD28, or a variant or homolog thereof, that maintains CD28 co-stimulatory domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity as compared to the CD28 co-stimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) as compared to a naturally occurring CD28 co-stimulatory domain polypeptide. In embodiments, the CD28 co-stimulatory domain is a human CD28 co-stimulatory domain protein. In embodiments, a variant or mutant of the CD28 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer deletions as compared to the naturally occurring CD28 co-stimulatory domain protein. In embodiments, a variant or mutant of the CD28 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions as compared to the naturally occurring CD28 co-stimulatory domain protein. In embodiments, a variant or mutant of the CD28 co-stimulatory domain protein contains no deletions as compared to the naturally occurring CD28 co-stimulatory domain protein. In embodiments, a variant or mutant of the CD28 co-stimulatory domain protein contains no insertions as compared to the naturally occurring CD28 co-stimulatory domain protein. In embodiments, a variant or mutant of the CD28 co-stimulatory domain protein contains substitutions that are conservative substitutions as compared to the naturally occurring CD28 co-stimulatory domain protein. In embodiments, the CD28 co-stimulatory domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001230006.1, or an isoform or naturally occurring variant or mutant thereof. In embodiments, the CD28 co-stimulatory domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001230007.1, or an isoform or naturally occurring variant or mutant thereof.In an embodiment, the CD28 co-stimulatory domain comprises all or part of a protein identified by NCBI sequence reference NP_006130.1, or an isoform or naturally occurring mutant or variant thereof.

[0057] As used herein, the term "4-1BB co-stimulatory domain" refers to either a co-stimulatory domain of 4-1BB or a recombinant or naturally occurring form of a variant or homolog thereof that maintains 4-1BB co-stimulatory domain activity (e.g., within a range of activity of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the 4-1BB co-stimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200-amino acid contiguous portion) compared to the naturally occurring 4-1BB co-stimulatory domain polypeptide. In embodiments, the 4-1BB co-stimulatory domain is a human 4-1BB co-stimulatory domain protein. In embodiments, a variant or mutant of the 4-1BB co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring 4-1BB co-stimulatory domain protein. In embodiments, a variant or mutant of the 4-1BB co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring 4-1BB co-stimulatory domain protein. In embodiments, a variant or mutant of the 4-1BB co-stimulatory domain protein contains no deletions compared to the naturally occurring 4-1BB co-stimulatory domain protein. In embodiments, a variant or mutant of the 4-1BB co-stimulatory domain protein contains no insertions compared to the naturally occurring 4-1BB co-stimulatory domain protein. In embodiments, a variant or mutant of the 4-1BB co-stimulatory domain protein contains substitutions that are conservative substitutions compared to the naturally occurring 4-1BB co-stimulatory domain protein. In embodiments, the amino acid sequence of the 4-1BB co-stimulatory domain protein has at least or about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 14.In an embodiment, the 4-1BB co-stimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001552.2, or an isoform or naturally occurring mutant or variant thereof.

[0058] As used herein, the term "ICOS co-stimulatory domain" refers to either a co-stimulatory domain of ICOS or a recombinant or naturally occurring form of a variant or homolog thereof that maintains ICOS co-stimulatory domain activity (e.g., within a range of activity that is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the ICOS co-stimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring ICOS co-stimulatory domain polypeptide. In embodiments, the ICOS co-stimulatory domain is a human "ICOS co-stimulatory domain protein. In embodiments, a variant or mutant of the ICOS co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring "ICOS co-stimulatory domain protein. In embodiments, a variant or mutant of the "ICOS co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring "ICOS co-stimulatory domain protein. In embodiments, a variant or mutant of the ICOS co-stimulatory domain protein contains no deletions compared to the naturally occurring ICOS co-stimulatory domain protein. In embodiments, a variant or mutant of the ICOS co-stimulatory domain protein contains no insertions compared to the naturally occurring ICOS co-stimulatory domain protein. In embodiments, a variant or mutant of the ICOS co-stimulatory domain protein contains substitutions that are conservative substitutions compared to the naturally occurring ICOS co-stimulatory domain protein. In embodiments, the ICOS co-stimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_036224.1, or an isoform or naturally occurring variant or mutant thereof.

[0059] As used herein, the term "OX-40 co-stimulatory domain" refers to either the co-stimulatory domain of OX-40 or a recombinant or naturally occurring form of a variant or homolog thereof that maintains OX-40 co-stimulatory domain activity (e.g., within a range of activity of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the OX-40 co-stimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200-amino acid segment) compared to the naturally occurring OX-40 co-stimulatory domain polypeptide. In embodiments, the OX-40 co-stimulatory domain is a human OX-40 co-stimulatory domain protein. In embodiments, a variant or mutant of the OX-40 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring OX-40 co-stimulatory domain protein. In embodiments, a variant or mutant of the OX-40 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring OX-40 co-stimulatory domain protein. In embodiments, a variant or mutant of the OX-40 co-stimulatory domain protein contains no deletions compared to the naturally occurring OX-40 co-stimulatory domain protein. In embodiments, a variant or mutant of the OX-40 co-stimulatory domain protein contains no insertions compared to the naturally occurring OX-40 co-stimulatory domain protein. In embodiments, a variant or mutant of the OX-40 co-stimulatory domain protein contains substitutions that are conservative substitutions compared to the naturally occurring OX-40 co-stimulatory domain protein. In embodiments, the OX-40 co-stimulatory domain comprises all or a portion of the protein identified by NCBI sequence reference NP_003318.1, or an isoform or naturally occurring variant or mutant thereof.

[0060] As used herein, the term "CTLA-4 co-stimulatory domain" refers to either the co-stimulatory domain of CTLA-4 or a recombinant or naturally occurring form of a variant or homolog thereof that maintains CTLA-4 co-stimulatory domain activity (e.g., within a range of activity of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the CTLA-4 co-stimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200 - amino acid portion) compared to the naturally occurring CTLA-4 co-stimulatory domain polypeptide. In embodiments, the CTLA-4 co-stimulatory domain protein is a human CTLA-4 co-stimulatory domain protein. In embodiments, the CTLA-4 co-stimulatory domain contains 5, 4, 3, 2, or 1 or fewer deletions. In embodiments, the CTLA-4 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions. In embodiments, the CTLA-4 co-stimulatory domain protein contains no deletions. In embodiments, the CTLA-4 co-stimulatory domain protein contains no insertions. In embodiments, the CTLA-4 co-stimulatory domain protein contains substitutions that are conservative substitutions. In embodiments, the CTLA-4 co-stimulatory domain is a human CTLA-4 co-stimulatory domain protein. In embodiments, a variant or mutant of the CTLA-4 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CTLA-4 co-stimulatory domain protein. In embodiments, a variant or mutant of the CTLA-4 co-stimulatory domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CTLA-4 co-stimulatory domain protein. In embodiments, a variant or mutant of the CTLA-4 co-stimulatory domain protein contains no deletions compared to the naturally occurring CTLA-4 co-stimulatory domain protein. In embodiments, a variant or mutant of the CTLA-4 co-stimulatory domain protein contains no insertions compared to the naturally occurring CTLA-4 co-stimulatory domain protein.In an embodiment, a variant or mutant of the CTLA-4 co-stimulatory domain protein comprises substitutions that are conservative substitutions compared to the naturally occurring CTLA-4 co-stimulatory domain protein.

[0061] As used herein, the term "CD3ζ intracellular T cell signaling domain" refers to either a recombinant or a naturally occurring form of the CD3ζ intracellular T cell signaling domain, or a variant or homolog thereof, that maintains CD3ζ intracellular T cell signaling domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD3ζ intracellular T cell signaling domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50-, 100-, 150-, or 200 - amino acid contiguous portion) compared to the naturally occurring CD3ζ intracellular T cell signaling domain polypeptide. In embodiments, the CD3ζ intracellular T cell signaling domain is a human CD3ζ intracellular T cell signaling domain protein. In embodiments, a variant or mutant of the CD3ζ intracellular T cell signaling domain protein contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, a variant or mutant of the CD3ζ intracellular T cell signaling domain protein contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, a variant or mutant of the CD3ζ intracellular T cell signaling domain protein contains no deletions compared to the naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, a variant or mutant of the CD3ζ intracellular T cell signaling domain protein contains no insertions compared to the naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, a variant or mutant of the CD3ζ intracellular T cell signaling domain protein contains substitutions that are conservative substitutions compared to the naturally occurring CD3ζ intracellular T cell signaling domain protein.In an embodiment, the CD3ζ intracellular T cell signaling domain comprises all or a portion of a protein specified by NCBI sequence reference NP_000725.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD3ζ intracellular T cell signaling domain comprises all or a portion of a protein specified by NCBI sequence reference NP_932170.1, or an isoform or naturally occurring mutant or variant thereof. Non-limiting examples of human CD3-zeta amino acid sequences available under the NCBI sequence references are specified above.

[0062] In an embodiment, the CD3ζ intracellular T cell signaling domain is encoded by all or part of a nucleic acid sequence identified by NCBI sequence reference NM_000734.3, or an isoform or naturally occurring variant or variant thereof. In an embodiment, the CD3ζ intracellular T cell signaling domain is encoded by all or part of a nucleic acid sequence identified by NCBI sequence reference NM_198053.2, or an isoform or naturally occurring variant or variant thereof. In an embodiment, a variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence contains 5, 4, 3, 2, or 1 or fewer deletions compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In an embodiment, a variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence contains 5, 4, 3, 2, or 1 or fewer insertions compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In an embodiment, a variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence does not contain deletions compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In an embodiment, a variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence does not contain insertions compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In an embodiment, a variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence contains substitutions that are conservative substitutions compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence.

[0063] A "control" sample or value refers to a sample that serves as a reference for comparison with a test sample, usually a known reference. For example, a test sample can be taken from test conditions in the presence of, for example, a test compound and can be compared to a sample from known conditions in the absence of, for example, the test compound (negative control) or in the presence of a known compound (positive control). A control can also represent an average value collected from several tests or results. One of ordinary skill in the art will recognize that a control can be designed for the evaluation of any number of parameters. For example, a control can be devised for comparing treatment effects based on pharmacological data (e.g., half-life) or treatment means (e.g., comparison of side effects). A control is also useful for determining the significance of data. For example, if the values of a given parameter vary widely in the control, the variation in the test sample is not considered significant.

[0064] As used herein, the terms "cancer" or "tumor" refer to all types of cancer, neoplasm, or malignant tumor found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated using the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that can be treated using the compounds or methods provided herein include thyroid cancer, endocrine cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, and uterine cancer. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma of the skin, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumor, malignant insulinoma, malignant carcinoid, bladder cancer, pre-malignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, hypercalcemia of malignancy, endometrial cancer, adrenocortical cancer, endocrine or exocrine pancreatic neoplasm, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular cancer, or prostate cancer.

[0065] As used herein (and well understood in the art), "treating" or "treatment" broadly encompasses any approach for obtaining a beneficial or desired result in the condition of a subject, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, whether partial or total and whether detectable or undetectable, reduction in the degree of a disease, stabilization of a medical condition (i.e., not worsening), prevention of infection or spread of a disease, delay or slowing of disease progression, improvement or alleviation of a medical condition, reduction of disease recurrence, and remission. In other words, "treatment" as used herein encompasses any cure, improvement, or prevention of a disease. Treatment can prevent the onset of a disease, inhibit the spread of a disease, alleviate the symptoms of a disease, completely or partially remove the underlying cause of a disease, shorten the duration of a disease, or any combination thereof.

[0066] As used herein, "treating" and "treatment" include prophylactic treatment. A treatment method includes administering a therapeutically effective amount of an active agent to a subject. The administration step can consist of a single administration or can include a series of administrations. The length of the treatment period depends on various factors such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will also be understood that the effective dosage of a drug used for treatment or prophylaxis can increase or decrease during a particular treatment or prophylaxis regimen. A change in dosage can be made, and this can be evident by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, treating or treatment is not prophylactic treatment.

[0067] "Patient" or "subject in need of treatment" refers to an organism that has or is susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the patient is human.

[0068] "Effective amount" means an amount of a compound that is sufficient to achieve a given objective (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition) compared to the absence of the compound. Examples of "effective amount" are amounts sufficient to contribute to the treatment, prevention, or reduction of symptoms of a disease, and may also be referred to as "therapeutically effective amount". "Reduction" of a symptom (and grammatical equivalents of this phrase) means a decrease in the severity or frequency of the symptom, or the elimination of the symptom. A "preventively effective amount" of a drug is the amount of the drug that, when administered to a subject, will have the intended preventive effect, e.g., prevent or delay the onset (or recurrence) of an injury, disease, disorder, or condition, or reduce the likelihood of the onset (or recurrence) of an injury, disease, lesion, or condition, or symptoms thereof. A complete preventive effect does not necessarily occur upon administration of a single dose and may occur only after administration of a series of doses. Thus, a preventively effective amount may be administered in one or more administrations. As used herein, "amount that reduces activity" refers to the amount of an antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. As used herein, "amount that interferes with function" refers to the amount of an antagonist required to interfere with the function of an enzyme or protein compared to the absence of the antagonist. The exact amount will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992), Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999), Pickar, Dosage Calculations (1999), and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0069] For any compound described herein, a therapeutically effective amount can first be determined from cell culture assays. The target concentration, when measured using the methods described herein or methods known in the art, will be the concentration of the active compound capable of achieving the methods described herein.

[0070] As is well known in the art, a therapeutically effective amount for use in humans can also be determined from animal models. For example, a human dosage can be formulated to achieve a concentration found to be effective in animals. The human dosage can be adjusted, as described above, by monitoring the effectiveness of the compound and adjusting the dosage up or down. Based on the above methods and other methods, adjusting the dosage to achieve maximum effectiveness in humans is well within the ability of one of ordinary skill in the art.

[0071] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to improve a disorder, as described above. For example, for a given parameter, a therapeutically effective amount will show at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% increase or decrease. Therapeutic effectiveness can also be expressed as an increase or decrease of "~-fold". For example, a therapeutically effective amount can be at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effective than a control.

[0072] Dosages can vary depending on the requirements of the patient and the compound being used. In light of the present disclosure, the dosage administered to a patient should be sufficient to provide a beneficial therapeutic response to the patient over time. The size of the dosage will also be determined by the presence, nature, and extent of any adverse side effects. Determining the appropriate dosage in a particular situation is within the ability of one of ordinary skill in the art. Generally, treatment is initiated at a dosage less than the optimal dosage of the compound. Thereafter, the dosage is increased gradually until the optimal effect is reached in the circumstances. Dosage and interval can be adjusted individually to provide a level of the administered compound effective for the particular clinical indication being treated. This will provide a treatment plan appropriate to the severity of the individual's medical condition.

[0073] As used herein, the term "administering" means administering to a subject by oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained release device, e.g., a mini-osmotic pump. Administration can be by any route, including parenteral and transmucosal (e.g., oral, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, arteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusions, transdermal patches, etc. In embodiments, administration does not include the administration of any active agent other than the recited active agents.

[0074] "Co-administering" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapeutic agents. The compounds provided herein can be administered to a patient either alone or co-administered. Co-administration is intended to include co-administration or sequential administration of the compounds, either individually or in combination (two or more compounds). Thus, the preparation can also be combined with other active substances, if desired (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally by a topical route or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0075] As used herein, the term "anti-cancer agent" refers to a molecule (e.g., a compound, peptide, protein, nucleic acid, 0103) used to treat cancer through the destruction or inhibition of cancer cells or tissues. An anti-cancer agent may be selective for a particular cancer or a particular tissue. The terms "Anti-cancer agent" and "anticancer agent" are used according to their plain and ordinary meaning and refer to a composition (e.g., a compound, drug, antagonist, inhibitor, modulator) having anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an agent identified herein that is useful in a method of treating cancer. In some embodiments, the anti-cancer agent is an agent approved by the FDA or a similar regulatory agency in a country other than the United States for treating cancer. Examples of anti-cancer agents include MEK (e.g., MEK1, MEK2, or both MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040, PD035901, selumetinib / AZD6244, GSK1120212 / trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uracil mustard, thiotepa, nitrosourea, nitrogen mustard (e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan), ethyleneimine and methylmelamine (e.g., hexamethylmelamine, thiotepa), alkyl sulfonate (e.g., busulfan), nitrosourea (e.g., carmustine, lomustine, semustine, streptozocin), triazene (dacarbazine)), antimetabolites (e.g., 5-azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analogs (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxuridine, cytarabine), purine analogs (e.g., mercaptopurine,Thioguanine, pentostatin), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum compounds (e.g., cisplatin, oxaliplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), adrenocortical suppressants (e.g., mitotane, aminoglutethimide), epipodophyllotoxin (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY43-9006, wortmannin, or LY294002, Syk inhibitor, mTOR inhibitor, antibodies (e.g., rituxan), gefitinib, gemtuzumab, polyphenol E, chlorofusin, all-trans retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all-trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec (registered trademark)), geldanamycin, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352, 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adesipenol, adozelesin, aldesleukin, ALL-TK antagonist, altretamine, ambamustine, amidozox, amifostine,Aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, dorsal dorsal formation protein-1, antiandrogen, prostate cancer, estrogen antagonist, anti-neoplastic drug, antisense oligonucleotide, aphidicolin glycinate, apoptosis gene modulator, apoptosis regulator, applinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, baranol, batimastat, BCR / ABL antagonist, benzochlorine, benzoyl staurosporine, beta-lactam derivative, beta-aretin, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinyl spermine, bisnafide, bistratene A, bizelesin, breflate, broxuridine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivative, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxamide triazole, CaRest M3, CARN 700, cartilage-derived inhibitor, calzerecin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analog, clotrimazole, corissamycin A, corissamycin B, combretastatin A4, combretastatin analog, conagenin, clambesidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentanequinone, cycloplatam, sipeamycin, cytarabine ocfosfate, cytolytic factor, cytostatic, daclizumab, decitabine, dehydradidemine B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemine B, didox, diethylnorspermine, dihydro-5-azacytidine, 9-dioxamycin, diphenylspirostatin,Docosanol, Dolasetron, Doxifluridine, Droloxifene, Dronabinol, Duocarmycin SA, Ebselen, Ecromycin, Edelfosine, Edrecolomab, Efrotomycin, Elemen, Emitefur, Epirubicin, Epristeride, Estramustine Analog, Estrogen Agonist, Estrogen Antagonist, Ethanidazole, Etoposide Phosphate, Exemestane, Fadrozole, Fludarabine, Fazarabine, Fenretinide, Filgrastim, Finasteride, Flavopiridol, Fleserastine, Fluasterone, Fludarabine, Fluorodaunorubicin Hydrochloride, Formestane, Fostriecin, Fotemustine, Gadolinium Texaphyrin, Gallium Nitrate, Gallocitabine, Ganirelix, Gelatinase Inhibitor, Gemcitabine, Glutathione Inhibitor, Hepsulfamide, Heregulin, Hexamethylene Bisacetamide, Hypericin, Ibandronic Acid, Idarubicin, Idoxifene, Idramantane, Ilmofosine, Iromastatin, Imidazoacridone, Imiquimod, Immunopotentiating Peptide, Insulin-like Growth Factor-1 Receptor Inhibitor, Interferon Agonist, Interferon, Interleukin, Iobenguane, Iododoxorubicin, Ipomoeanol, 4-, Irinotecan, Iroplact, Isogladine, Isobenzazole, Isohomohalicondrin B, Itasetron, Jasplakinolide, Kahalalide F, Lamelarin-N Triacetate, Lanreotide, Rayamycin, Lenograstim, Lentinin Sulfate, Leptostatin, Letrozole, Leukemia Inhibitory Factor, Leukocyte Alpha Interferon, Leuprorelin + Estrogen + Progesterone, Leuprolide, Levamisole, Rialoxazole, Linear Polyamine Analog, Liposoluble Disaccharide Peptide, Liposoluble Platinum Compound, Lysoclinamide 7, Lobaplatin, Lombricin, Lometrexol, Lonidamine, Losoxantrone, Lovastatin, Roxoribine, Lurtotecan, Lutetium Texaphyrin, Lysophylline, Lytic Peptide, Maytansine, Mannostatin A, Marimastat, Masoprocol, Maspin, Matrilysin Inhibitor, Matrix Metalloproteinase Inhibitor, Menogaril, Melvalone, Meteleren,Methioninase, Metoclopramide, MIF inhibitor, Mifepristone, Miltefosine, Milomostim, Mismatch double-stranded RNA, Mitoguazone, Mitolactol, Mitomycin analog, Mitonafide, Mitotoxin fibroblast growth factor-Saporin, Mitoxantrone, Mofarotene, Molgramostim, Monoclonal antibody, Human chorionic gonadotropin, Monophosphoryl lipid A + Mycobacterium cell wall sk, Mopidamol, Multidrug resistance gene inhibitor, Multiple tumor suppressor 1-based therapeutic agent, Mustard anticancer agent, Mycoparoxide B, Mycobacterium cell wall extract, Miliapolone, N-acetyl dinarine, N-substituted benzamide, Naphthalene, Nagrestip, Naloxone + Pentazocine, Napabine, Naftelpin, Nartograstim, Nedaplatin, Nemorubicin, Neridronic acid, Neutral endopeptidase, Nilutamide, Nisamycin, Nitric oxide modulator, Nitroxide antioxidant, Nitrilin, O6-benzylguanine, Octreotide, Oxenone, Oligonucleotide, Onapristone, Ondansetron, Ondansetron, Orasin, Oral cytokine inducer, Ormaplatin, Osaterone, Oxaliplatin, Oxazunomycin, Paraureamine, Palmitoyl lysophosphatidylcholine, Pamidronic acid, Panaxytriol, Panomifene, Parabactin, Pazelliptine, Pegaspargase, Perdesin, Pentosan polysulfate sodium, Pentostatin, Pentolozole, Perflubron, Perfosfamide, Perillyl alcohol, Phenazinomycin, Phenyl acetate, Phosphatase inhibitor, Picibanil, Pilocarpine hydrochloride, Pirarubicin, Pyrithioxine, Placetin A, Placetin B, Plasminogen activator inhibitor, Platinum complex, Platinum compound, Platinum-triamine complex, Porfimer sodium, Porfiromycin, Prednisone, Propylbis-acridone, Prostaglandin J2, Proteasome inhibitor, Protein A-based immunomodulator, Protein kinase C inhibitor, Protein kinase C inhibitor, Microalgae, Protein tyrosine phosphatase inhibitor, Purine nucleoside phosphorylase inhibitor, Purpurin, Pyrazoloacridine, Pyridoxylated hemoglobin polyoxyethylene conjugate, Raf antagonist, Raltitrexed,Lamotrigine, ras farnesyl protein transferase inhibitor, ras inhibitor, ras-GAP inhibitor, demethylated leteprinim, rhenium etidronate Re 186, lysokin, ribozyme, RII retinamide, logretimide, rohitukine, romurtide, Rokinimex, Rubiginone B1, Ruboxyl, Saffingol, Syntopin, SarCNU, Sarcophytol A, Sargramostim, Sdi 1 mimetic, Semustine, Aging-derived inhibitor 1, Sense oligonucleotide, Signal transduction inhibitor, Signal transduction modulator, Single-chain antigen-binding protein, Schizophyllan, Sobuzoxane, Borocaptate sodium, Sodium phenylacetate, Solverol, Somatomedin-binding protein, Sonermin, Sparfosic acid, Spicamycin D, Spiro-mustin, Sprenopentin, Spongistatin 1, Squaramine, Stem cell inhibitor, Stem cell division inhibitor, Stipiamide, Stromelysin inhibitor, Sulfinosine, Superactive vasoactive intestinal peptide antagonist, Suradista, Slamine, Swainsonine, Synthetic glycosaminoglycan, Talimustine, Tamoxifen methiodide, Tauromustine, Tazarotene, Tegafur sodium, Tegafur, Terlapirium, Telomerase inhibitor, Temoporfin, Temozolomide, Teniposide, Tetrachlorodecaoxide, Tetrazomine, Salibrastin, Thiocholic acid, Thrombopoietin, Thrombopoietin mimetic, Thymalfasin, Thymopoietin receptor agonist, Timotrinan, Thyroid-stimulating hormone, Tin ethyl ethiopurpurin, Tirapazamine, Titanocene dichloride, Topseptin, Toremifene, Totipotent stem cell factor, Translation inhibitor, Tretinoin, Triacetyluridine, Trisciribine, Trimethotrexate, Triptorelin, Tropisetron, Tursteride, Tyrosine kinase inhibitor, Tyrphostin, UBC inhibitor, Ubenimex, Urogenital sinus-derived growth inhibitor, Urokinase receptor antagonist, Bapreotide, Variolin B, Vector system, Erythrocyte gene therapy agent, Veraresol, Veramine, Verdins, Verteporfin, Vinorelbine, Vincaltine, Vitaxin, Borozole, Zanoterone, Zeniplatin, Dirascolb, Dinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, Asibiicin, Aclarubicin, Hydrochloride acodazole, Acronine, Adzelesin, Aldesleukin, Altretamine, Ambomycin, Amethopterin acetate,Aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, broxuridine, busulfan, calicheamicin, caracemide, carbetimer, carboplatin, carmustine, carboquone hydrochloride, carzelesin, cerulenin, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, daunorubicin hydrochloride, decitabine, dexormaplatin, desaguanine, desaguanine mesylate, diaziquone, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drostanolone propionate, duazomycin, edatrexate, efloxatin hydrochloride, elsamitrucin, enloplatin, empromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocytosine, fosquidone, phosphotriesin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, iimofosine, interleukin I1 (including recombinant interleukin II or rlL.sub.2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-1a, interferon gamma-1b, iplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, methopterin, metsuldepa,Mitindomide, Mitocarcin, Mitocromin, Mitogillin, Mitomalcin, Mitomycin, Mitosper, Mitotan, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole, Nogalamycin, Oxaliplatin, Oxisuran, Pegaspargase, Peplomycin Sulfate, Perfosfamide, Pipobroman, Piposulfan, Pyroxantrone Hydrochloride, Plicamycin, Promestane, Porfimer Sodium, Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin, Puromycin Hydrochloride, Pyrazofurin, Rizoxin, Logretimide, Safingol, Safingol Hydrochloride, Semustine, Simtrazene, Sparfosate Sodium, Sparsomycin, Spirogermanium Hydrochloride, Spiroplatin, Streptozocin, Streptonigrin, Streptozocin, Thiotepa, Thiazofurin, Tiraparazamine, Toremifene Citrate, Trestolone Acetate, Trisciribine Phosphate, Trimethoprim, Trimethoprim Glucuronate, Triptorelin, Tebuconazole Hydrochloride, Uracil Mustard, Uredepa, Bapreotide, Verteporfin, Vinblastine Sulfate, Vincristine Sulfate, Vindesine, Vindesine Sulfate, Vinepidine Sulfate, Vincoglycinate Sulfate, Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine Sulfate, Vinzolidine Sulfate, Borozole, Zeniplatin, Dinostatin, Zorubicin Hydrochloride, Agents that arrest cells in G2-M phase and / or modulate microtubule formation or stability (e.g., Taxol (trademark) (i.e., paclitaxel), Taxotere (trademark), compounds containing the taxane backbone, Elbuzole (i.e., R-55104), Drastatin 10 (i.e., DLS-10 and NSC-376128), Mivobulin Isethionate (i.e., as CI-980), Vincristine, NSC-639829, Discodermolide (i.e., as NVP-XX-A-296), ABT-751 (Abbott, i.e., E-7010), Altretamine (e.g.,Altorticin A and Altorticin C), Spongistatin (e.g., Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Semadotin Hydrochloride (i.e., LU-103793 and NSC-D-669356), Epothilone (e.g., Epothilone A, Epothilone B, Epothilone C (i.e., Deoxyepothilone A or dEpoA), Epothilone D (i.e., KOS-862, dEpoB, and Deoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-Aza-epothilone B, 21-Aminoepothilone B (i.e., BMS-310705), 21-Hydroxyepothilone D (i.e., Deoxyepothilone F and dEpoF), 26-Fluoroepothilone, Auristatin PE (i.e., NSC-654663), Soblidotin (i.e., TZT-1027), LS-4559-P (Pharmacia, i.e., LS-4577), LS-4578 (Pharmacia, i.e., LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine Sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e., WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e., ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e., LY-355703), AC-7739 (Ajinomoto, i.e., AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e., AVE-8062, AVE-8062A,CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e., NSC-106969), T-138067 (Tularik, i.e., T-67, TL-138067, and TI-138067), COBRA-1 (Parker Hughes Institute, i.e., DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (i.e., BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Physanolide B, Laurimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e., SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-569), Narcosin (also known as NSC-5366), Noscapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanosine (i.e., NSC-698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e., T-900607), RPR-115781 (Aventis), Erythrobins (such as desmethylerythrobins, desaetyleleutherobin, isoerythrobins A, and Z-erythrobins), Calycoside, Calycosin, Halicondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonaamide A, A-293620 (Abbott), NPI-2350 (Nereus), Takalonolide A,TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-phenylahistin (i.e., NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), myoseverin B, D, -43411 (Zentaris, i.e., D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e., SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin sodium phosphate, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH), e.g., goserelin or leuprolide, corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethylstilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111 In, 90 Y, or 131Anti-CD20 monoclonal antibody conjugated to I, etc., tripterid, homoharringtonin, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, seribastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitor, epidermal growth factor receptor (EGFR) targeted therapy or therapeutic agent (e.g., gefitinib (Iressa (trademark)), erlotinib (Tarceva (trademark)), cetuximab (Erbitux (trademark)), lapatinib (Tykerb (trademark)), panitumumab (Vectibix (trademark)), vandetanib (Caprelsa (trademark)), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, etc. are mentioned, but not limited thereto.

[0076] "Selective" or "selectivity" of a compound refers to the ability of the compound to distinguish a molecular target (e.g., a compound having selectivity for ROR1).

[0077] "Specific", "specifically", "specificity" of a compound refers to the ability of the compound to cause a specific action such as inhibition on a specific molecular target with minimal or no action on other proteins in the cell.

[0078] "ROR1 inhibitor" refers to a compound (e.g., a compound described herein) that reduces the activity of ROR1 as compared to a control such as a compound absent or having known inactivity.

[0079] "Contacting" is used in accordance with its plain and ordinary meaning and refers to a process that enables at least two different species (e.g., compounds including biomolecules or cells) to react, interact, or be in close proximity sufficient to physically contact. However, it should be understood that the resulting reaction product can be generated directly from the reaction between the added reagents or from intermediates derived from one or more of the added reagents that can be generated in the reaction mixture.

[0080] As defined herein, terms such as "inhibit", "inhibiting", "inhibition", etc. with respect to protein-inhibitor interactions mean to adversely affect (e.g., reduce) the activity or function of a protein as compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition means to have a negative effect (e.g., reduce) on the protein concentration or level as compared to the protein concentration or level in the absence of the inhibitor. In embodiments, inhibition refers to the alleviation of a disease or symptoms of a disease. In embodiments, inhibition refers to a decrease in the activity of a specific protein target. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, decreasing signal transduction or enzyme activity or the amount of a protein, preventing, or delaying activation, or inactivating, desensitizing, or downregulating. In embodiments, inhibition refers to a decrease in the activity of a target protein due to a direct interaction (e.g., the inhibitor binds to the target protein). In embodiments, inhibition refers to a decrease in the activity of a target protein due to an indirect interaction (e.g., the inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein). A "ROR1 inhibitor" is a compound that adversely affects (e.g., reduces) the activity or function of ROR1 as compared to the activity or function of ROR1 in the absence of the inhibitor.

[0081] The term "expression" includes any stage involved in polypeptide production, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0082] In the context of a substance or the activity or function of a substance related to a disease (e.g., a protein-related disease, a cancer related to ROR1 activity, an ROR1-related cancer, an ROR1-related disease (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)), the terms "related" or "associated with" mean that the disease (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease) is (wholly or in part) caused by, or the symptoms of the disease are (wholly or in part) caused by the substance or the activity or function of the substance. As used herein, something described as related to a disease can be a target for treating the disease if it is a causative agent. For example, if an increase in ROR1 activity or function (e.g., signaling pathway activity) causes a disease (e.g., cancer, inflammatory disease, autoimmune disease, or infection), a cancer or ROR1-related disease (e.g., cancer, inflammatory disease, autoimmune disease, or infection) related to ROR1 activity or function can be treated with an ROR1 modulator or an ROR1 inhibitor. For example, if an increase in ROR1 activity or function (e.g., signaling pathway activity) causes a disease, an inflammatory disease or an ROR1-related inflammatory disease related to ROR1 activity or function can be treated with an ROR1 modulator or an ROR1 inhibitor.

[0083] As used herein, the term "signaling pathway" refers to a series of interactions between cellular components and any extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) in which a change in one component is transmitted to one or more other components, which then transmit the change to additional components, which is optionally propagated to other signaling pathway components. The examples and embodiments described in this specification are for illustrative purposes only, and various modifications or changes in light of them will be suggested to those skilled in the art and should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0084] Composition Provided herein are, inter alia, recombinant protein / chimeric antigen receptor (these terms are used interchangeably throughout) compositions useful for the treatment of hematological malignancies, and methods of using the same. Applicants have discovered that chimeric antigen receptors (CARs) directed to ROR-1 provide highly active and efficient immunotherapy compositions. Without being bound by a particular theory, antibodies that are known to inhibit the receptors to which they bind and downregulate their surface expression are generally not considered to be good clinical candidates for CARs. Thus, it was very surprising that CARs comprising the CDRs of the ROR-1 antibodies described herein, including embodiments thereof, exhibit effective cancer-specific cytotoxicity when expressed by T cells.

[0085] In one aspect, a recombinant protein or a composition comprising a recombinant protein is provided, which comprises an antigen-binding region, preferably an ROR-1 binding domain, and a transmembrane domain. In embodiments, the ROR-1 binding domain is an antibody. In embodiments, the ROR-1 binding domain is an antibody fragment.

[0086] In one aspect, a recombinant protein is provided, which comprises (i) an antibody region comprising (a) a light chain variable domain comprising CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and (b) a heavy chain variable domain comprising CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, and (ii) a transmembrane domain.

[0087] In one aspect, a chimeric antigen receptor is provided. The chimeric antigen receptor includes: i. an antigen-binding region that specifically binds to ROR-1, a transmembrane domain, a spacer domain that binds the antigen-binding region and the transmembrane domain, and an intracellular domain. The terms "antibody region", "antigen-binding region", or "antigen-binding domain" provided herein are used interchangeably throughout and refer to a monovalent or multivalent protein moiety (i.e., a recombinant protein, chimeric antigen receptor) that forms part of the proteins provided herein, including its embodiments. Thus, one of ordinary skill in the art will immediately recognize that an antibody region or antigen-binding region is a protein moiety capable of binding to an antigen (epitope). In embodiments, the ROR-1 binding domain includes an antibody region comprising a light chain variable region (V L ) and a heavy chain variable region (V H ). In embodiments, the light chain variable domain includes CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain includes CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48. It should be noted that these CDRs are those of, or derived from, siltuximab (also known as UC-961 or 99961.1). The development and structure of siltuximab are disclosed in U.S. Patent Application No. 14 / 422,519, which is hereby incorporated by reference in its entirety for all purposes. In embodiments, the ROR-1 light chain variable domain includes CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain includes CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54.

[0088] In one aspect, a chimeric antigen receptor is provided. The chimeric antigen receptor comprises: i. an antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1, the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain, and (a) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54; ii. a spacer domain comprising a spacer having a length of 10 to 240 amino acids; iii. a transmembrane domain; and iv. an intracellular domain.

[0089] In one aspect, a chimeric antigen receptor is provided. The chimeric antigen receptor comprises: i. an antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1, the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain, and (a) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54; an antigen-binding region; ii. a transmembrane domain; and iii. an intracellular domain comprising an intracellular T cell signaling domain and an intracellular co-stimulatory domain selected from 4-1BB, ICOS, OX-40, and combinations thereof.

[0090] As used interchangeably throughout this specification, the terms "antibody region" and "antigen-binding region" refer to a monovalent or multivalent protein moiety (i.e., a recombinant protein, a chimeric antigen receptor) that forms part of a protein provided herein, including embodiments thereof. Thus, one of ordinary skill in the art will immediately recognize that an antibody region or antigen-binding region is a protein moiety capable of binding to an antigen (epitope). The antibody regions presented herein may include domains of an antibody or fragments thereof (e.g., Fab). Thus, an antibody region may include a light chain variable domain (VL) and / or a heavy chain variable domain (VH).

[0091] The provided light chain variable (VL) domain comprises the CDR sequences and framework region (FR) sequences of the light chain of an antibody, antibody variant, or fragment thereof. In embodiments, the antibody region or antigen-binding region comprises a variable light chain domain and a variable heavy chain domain. As used herein, "variable light chain domain" refers to a polypeptide contained within (and forming part of) the variable light chain (VL) region. In embodiments, the variable light chain region is the light chain variable (VL) domain. As used herein, "variable heavy chain domain" refers to a polypeptide contained within (and forming part of) the variable heavy chain (VH) region. In embodiments, the variable heavy chain region is the heavy chain variable (VH) domain. In embodiments, the light chain variable (VL) domain comprises CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45). In embodiments, the heavy chain variable (VH) domain comprises CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48). It should be noted that these CDRs are those of, or derived from, siltuximab (also known as UC-961 or 99961.1). The development and structure of siltuximab are disclosed in U.S. Patent Application No. 14 / 422,519, which is hereby incorporated by reference in its entirety for all purposes.

[0092] In an embodiment, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21. In an embodiment, the light chain variable domain has the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21. In an embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27. In an embodiment, the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27. In an embodiment, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19. In an embodiment, the light chain variable domain has the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19. In an embodiment, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20. In an embodiment, the light chain variable domain has the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20. In an embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25. In an embodiment, the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25. In an embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 26. In an embodiment, the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 26.

[0093] In an embodiment, the C-terminus of the light chain variable domain is bound to the N-terminus of the heavy chain variable domain. In an embodiment, the N-terminus of the light chain variable domain is bound to the C-terminus of the heavy chain variable domain. In an embodiment, the light chain variable domain is covalently bound to the heavy chain variable domain via a chemical linker. The "chemical linker" provided herein is a covalent linker, a non-covalent linker, a peptide linker (a linker containing a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, or any combination thereof. Thus, a chemical linker as provided herein may contain multiple chemical moieties, where each of the multiple chemical moieties is chemically distinct. Alternatively, the chemical linker may be a non-covalent linker. Examples of non-covalent linkers include, but are not limited to, ionic bonds, hydrogen bonds, halogen bonds, van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi-effects), and hydrophobic interactions. In an embodiment, the chemical linker is formed using conjugate chemistry including, but not limited to, nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). In an embodiment, the chemical linker is a peptide linker having a length of 5 to 100, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 10 to 50, 10 to 40, or 10 to 30 amino acids. Any sequence that provides sufficient flexibility between the light chain variable domain and the heavy chain variable domain is contemplated for the recombinant proteins provided herein, including that embodiment. In an embodiment, the peptide linker comprises, or has, the amino acid sequence of SEQ ID NO: 24. In an embodiment, the peptide linker has an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 24.

[0094] Any transmembrane domain capable of immobilizing the proteins provided herein, including that embodiment, is contemplated. Any suitable transmembrane domain is contemplated herein, and exemplary but non-limiting examples of transmembrane domains include the transmembrane domains of CD28, CD8, CD4, CD3ζ, or CD8α. In a preferred embodiment, the transmembrane domain is the CD28 transmembrane domain. As used herein, the term "CD28 transmembrane domain" refers to either a recombinant or naturally occurring form of the transmembrane domain of CD28 or a variant or homolog thereof that maintains CD28 transmembrane domain activity (e.g., within a range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD28 transmembrane domain). In some embodiments, the variant or homolog has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity across the entire sequence or a portion of the sequence of the naturally occurring CD28 transmembrane domain polypeptide. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD28 transmembrane domain is the amino acid sequence of SEQ ID NO: 32. In an embodiment, the CD28 transmembrane domain in the ROR1 CAR described herein comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.

[0095] In an embodiment, the C-terminus of the heavy chain variable domain is bound to the N-terminus of the transmembrane domain. In an embodiment, the C-terminus of the light chain variable domain is bound to the N-terminus of the transmembrane domain. In an embodiment, the heavy chain variable domain or the light chain variable domain is covalently bound to the transmembrane domain through a spacer domain. In an embodiment, the binding affinity of the antigen-binding region for the antigen is increased in a CAR construct having a spacer domain. In an embodiment, the activity of a CAR having a spacer domain is higher than that of a CAR without a spacer domain. In an embodiment, the flexibility of the antigen-binding region of the CAR is increased by the presence of the spacer domain.

[0096] Any suitable chemical or biological moiety that can provide space or flexibility to the antigen-binding region or antigen-binding domain without providing excessive steric hindrance to the antigen-binding domain is contemplated. Preferably, the spacer domain comprises a polypeptide having a length of 10 to 250 amino acids, 12 to 250 amino acids, 12 to 200 amino acids, 14 to 250 amino acids, 14 to 200 amino acids, 14 to 150 amino acids. Accordingly, exemplary spacer domains herein include immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4, or the constant heavy chain 1 (CH1), constant heavy chain 2 (CH2), or constant heavy chain 3 (CH3) domains of IgG1, IgG2, IgG3, or IgG4), or immunoglobulin molecules or fragments thereof that contain mutations affecting Fc receptor binding (e.g., IgG1, IgG2, IgG3, IgG4). Alternatively and / or additionally, some contemplated spacer domains include all or part of the hinge region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4), i.e., the sequence located between the CH1 and CH2 domains of the immunoglobulin, such as the IgG4 Fc hinge or the CD8 hinge. Some spacer domains include the immunoglobulin CH3 domain, or both the CH3 and CH2 domains. Immunoglobulin-derived sequences may include one or more amino acid modifications, such as 1, 2, 3, 4, or 5 substitutions (e.g., substitutions that reduce off-target binding) or additions of less than 10, less than 5, or less than 3 amino acids at its N-terminus or C-terminus.

[0097] In embodiments, the spacer domain comprises a hinge domain and optionally one or both of the CH3 and CH2 domains of an immunoglobulin molecule. Thus, in embodiments, the spacer domain has or consists of a hinge region or a modified hinge region (e.g., with one or more amino acid substitutions or additions). In embodiments, the spacer domain has or consists of a hinge region that is bound to the CH3 domain of hIgG4 at the C-terminus of the hinge region. In embodiments, the spacer domain has or consists of a hinge region that is bound to the CH2 domain of hIgG4 at the C-terminus of the hinge region. Alternatively, the spacer domain has or consists of a hinge region that is bound to the CH2-CH3 domain of hIgG4 at the C-terminus of the hinge region. In embodiments, the hinge region as used herein comprises or consists of the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29. In embodiments, the spacer domain comprises the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42. In embodiments, the spacer domain has or consists of the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0098] In embodiments, the recombinant protein (e.g., the intracellular domain) further comprises one or more intracellular co-stimulatory signaling domains. As used herein, an "intracellular co-stimulatory signaling domain" includes an amino acid sequence that can provide co-stimulatory signaling in response to binding of an antigen to an antibody region provided herein, including embodiments thereof. In embodiments, the signaling of the co-stimulatory signaling domain results in cytokine production and proliferation of T cells expressing the same. In embodiments, the intracellular co-stimulatory signaling domain includes at least one or more of the CD28 intracellular co-stimulatory signaling domain, the 4-1BB (CD137) intracellular co-stimulatory signaling domain, the ICOS intracellular co-stimulatory signaling domain, the OX-40 intracellular co-stimulatory signaling domain, or any combination thereof. For example, a preferred intracellular co-stimulatory signaling domain comprises or consists of at least a portion of the CD28 intracellular co-stimulatory signaling domain, or at least a portion of the 4-1BB (CD137) intracellular co-stimulatory signaling domain. Another preferred intracellular co-stimulatory signaling domain comprises or consists of at least a portion of the CD28 intracellular co-stimulatory signaling domain bound to at least a portion of the 4-1BB (CD137) intracellular co-stimulatory signaling domain. In embodiments, at least a portion of the CD28 intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In embodiments, at least a portion of the CD28 intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In embodiments, at least a portion of the 4-1BB intracellular co-stimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 33.In an embodiment, at least a part of the 4-1BB intracellular co-stimulatory signaling domain comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29. In an embodiment, the chimeric antigen receptor or recombinant protein disclosed herein is a combination of SEQ ID NO: 32 and SEQ ID NO: 33 (either SEQ ID NO: 32 is present at the N'-terminus of SEQ ID NO: 33 or SEQ ID NO: 33 is present at the N'-terminus of SEQ ID NO: 32), or a combination of SEQ ID NO: 32 and SEQ ID NO: 33 (either SEQ ID NO: 32 is present at the N'-terminus of SEQ ID NO: 33 or SEQ ID NO: 33 is present at the N'-terminus of SEQ ID NO: 32) and an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical thereto.

[0099] In an embodiment, the recombinant protein further comprises an intracellular T cell signaling domain. As used herein, an "intracellular T cell signaling domain" includes an amino acid sequence that can provide primary signaling in response to binding of an antigen to an antibody region provided herein, including embodiments thereof. In an embodiment, signaling by the intracellular T cell signaling domain results in activation of the T cells expressing it. In an embodiment, signaling by the intracellular T cell signaling domain results in proliferation (cell division) of the T cells expressing it. In an embodiment, signaling by the intracellular T cell signaling domain results in expression by T cells of proteins known in the art that are characteristic of activated T cells (e.g., CTLA-4, PD-1, CD28, CD69). In an embodiment, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain. In an embodiment, the CD3ζ intracellular T cell signaling domain comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34 and that generates a CD3ζ intracellular T cell signaling domain having at least 80%, 85%, 90%, or 95% of the activity of the CD3ζ intracellular T cell signaling domain having SEQ ID NO: 34. In an embodiment, the CD3ζ intracellular T cell signaling domain has an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34 and that generates a peptide having at least 80%, 85%, 90%, or 95% of the activity of the CD3ζ intracellular T cell signaling domain having SEQ ID NO: 34.

[0100] In an embodiment, the ROR-1 CAR disclosed herein comprises i) an ROR1 scFv (a sequence of SEQ ID NO: 21 having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a sequence of SEQ ID NO: 27, or a VH domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv (CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or a sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a sequence of SEQ ID NO: 20, or a VH domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), ii) a spacer domain having a hinge and CH2 and CH3 domains, iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or consists of or comprises them.

[0101] Alternatively, in an embodiment, the ROR1 CAR disclosed herein is i) an ROR1 scFv (having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), the sequence of SEQ ID NO: 21, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv (CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), and ii) a spacer domain having a hinge and CH2 domains, and iii) a CD28 transmembrane domain, and iv) a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or comprises or consists of these.

[0102] Alternatively, in an embodiment, the ROR1 CAR disclosed herein is i) an ROR1 scFv (having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), the sequence of SEQ ID NO: 21, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv (CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), ii) a spacer domain having a hinge and CH3 domains, iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or comprises or consists of them.

[0103] Alternatively, in an embodiment, the ROR1 CAR disclosed herein is i) an ROR1 scFv having the sequence of SEQ ID NO: 21 having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27, or alternatively, an ROR1 scFv having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20, and ii) a spacer domain having a hinge and a portion of the CH3 domain (e.g., half of the CH3 domain, 43 amino acids at the N-terminus of the CH3 domain, etc.), iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or comprises or consists of them.

[0104] Alternatively, in an embodiment, the ROR1 CAR disclosed herein is i) an ROR1 scFv having the sequence of SEQ ID NO: 21 having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), and ii) a spacer domain having a hinge domain, iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or comprises or consists of them.

[0105] As a result, in an embodiment, the ROR1 CAR disclosed herein comprises: i) an ROR1 scFv (a sequence of SEQ ID NO: 21 having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or a VL domain having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a sequence of SEQ ID NO: 27, or a VH domain having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv (CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or a sequence of SEQ ID NO: 19, or a VL domain having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a sequence of SEQ ID NO: 20, or a VH domain having a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), ii) a spacer domain having a hinge and CH2 and CH3 domains, iii) a CD28 transmembrane domain, iv) a CD28 co-stimulatory domain bound to a 4-1BB (CD137) co-stimulatory domain, and v) a CD3Z T cell activation domain, or consists of these components.

[0106] Alternatively, in an embodiment, the ROR-1 CAR disclosed herein is i) an ROR1 scFv (having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), the sequence of SEQ ID NO: 21, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27), or alternatively, an ROR1 scFv (having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20), ii) a spacer domain having a hinge and CH2 domains, iii) a CD28 transmembrane domain, iv) a CD28 co-stimulatory domain bound to a 4-1BB (CD137) co-stimulatory domain, and v) a CD3 zeta cell activation domain, or consists of or comprises them.

[0107] Alternatively, in an embodiment, the ROR-1 CAR disclosed herein is i) an ROR1 scFv having the sequence of SEQ ID NO: 21, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27, having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or alternatively, an ROR1 scFv having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20, and ii) a spacer domain having a hinge and CH3 domains, and iii) a CD28 transmembrane domain, and iv) a CD28 co-stimulatory domain linked to a 4-1BB (CD137) co-stimulatory domain, and v) a CD3ζ T cell activation domain, or comprises or consists of these.

[0108] Alternatively, in an embodiment, the ROR1 CAR disclosed herein is: i) an ROR1 scFv having the sequence of SEQ ID NO: 21 having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27, or alternatively, an ROR1 scFv having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20; ii) a spacer domain having a hinge and a portion of the CH3 domain (e.g., half of the CH3 domain, 43 amino acids at the N-terminus of the CH3 domain, etc.); iii) a CD28 transmembrane domain; iv) a CD28 co-stimulatory domain linked to a 4-1BB (CD137) co-stimulatory domain; and v) a CD3Z T cell activation domain, or consists of or comprises these components.

[0109] Alternatively, in an embodiment, the ROR-1 CAR disclosed herein comprises i) an ROR1 scFv having the sequence of SEQ ID NO: 21, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21 and a VH domain having the sequence of SEQ ID NO: 27, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27, and having CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45), CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48), or alternatively, an ROR1 scFv having CDR L1 (SEQ ID NO: 49), CDR L2 (SEQ ID NO: 50), and CDR L3 (SEQ ID NO: 51), CDR H1 (SEQ ID NO: 52), CDR H2 (SEQ ID NO: 53), and CDR H3 (SEQ ID NO: 54), or the sequence of SEQ ID NO: 19, or a VL domain having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19 and a VH domain having the sequence of SEQ ID NO: 20, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20, and ii) a spacer domain having a hinge domain, iii) a CD28 transmembrane domain, iv) a CD28 co-stimulatory domain bound to a 4-1BB (CD137) co-stimulatory domain, and v) a CD3ζ T cell activation domain, or consists of these components.

[0110] The chimeric antigen receptor described herein is intended to bind to a peptide containing glutamic acid at a position corresponding to amino acids 130-160 of ROR-1 or a fragment thereof, preferably at position 138 of the ROR-1 polypeptide. Alternatively and / or additionally, the chimeric antigen receptor described herein specifically binds to either the 3' or middle Ig-like region of the extracellular domain of the ROR-1 protein, preferably to the 3' end of the Ig-like region of the extracellular domain of the ROR-1 protein at positions 1-147.

[0111] As a result, the ROR-1 CAR disclosed in this specification can bind to ROR-1-expressing cells, including cancer cells such as leukemia cells, lymphoma cells, chronic lymphocytic leukemia (CLL) cells, adult acute myeloid leukemia (AML) cells, acute lymphoblastic leukemia (ALL) cells, mantle cell lymphoma cells, ovarian cancer cells, colon cancer cells, lung cancer cells, skin cancer cells, pancreatic cancer cells, testicular cancer cells, bladder cancer cells, uterine cancer cells, prostate cancer cells, breast cancer cells, or adrenal cancer cells, and initiate or induce an immune response against the ROR-1-expressing cells, and this recombinant protein forms part of the cell.

[0112] In embodiments, including that embodiment, the recombinant proteins provided herein further include a detectable domain. As used herein, a "detectable domain" is a peptide moiety detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, a detectable domain provided herein can be a protein or other entity that can be made detectable, for example, by incorporating a radiolabel or by being specifically reactive with an antibody. Any suitable method known in the art for conjugating an antibody to a label may be used (e.g., the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego). In the present invention, the detectable domain is used to confirm transfection of T cells.

[0113] In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 500 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 550 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 600 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 650 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 700 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 750 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 800 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 850 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 900 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 950 pM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1.5 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2.5 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3.5 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4.5 nM to about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5 nM to about 6 nM.In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5.5 nM to about 6 nM.

[0114] In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 500 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 500 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 550 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 550 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 600 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 600 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 650 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 650 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 700 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 700 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 750 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 750 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 800 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 800 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 850 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 850 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 900 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 900 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 950 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 950 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM.In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 1 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 1.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 2 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 2.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 3 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 3.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 4 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 4.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 5.5 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of 6 nM.

[0115] In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 40 nM (e.g., 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 nM). D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 40 nM (e.g., 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 nM). D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 35 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 35 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 30 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 30 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 25 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 25 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 20 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 20 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 15 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than 15 nM. D In an embodiment, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a K of less than about 10 nM. Dand binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 10 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 9 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 9 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 8 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 8 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 7 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 7 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 6 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 6 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 5 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 5 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 4 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 4 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 3 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 3 nM Dand binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 2 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 2 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 1 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 1 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 0.5 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 0.5 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 0.25 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 0.25 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than about 0.1 nM D and binds to the ROR-1 protein. In embodiments, the recombinant protein or chimeric antigen receptor has a K of less than 0.1 nM D and binds to the ROR-1 protein.

[0116] The chimeric antigen receptors described herein are intended to be expressed on the surface of T lymphocytes or T cells (e.g., CD8+ T cells, CD4+ T cells) by genetically engineering T cells to express a heterologous nucleic acid sequence encoding a chimeric nucleic acid receptor. Such generated CAR-T cells specifically bind to ROR-1 expressing cells, preferably ROR1-expressing cancer cells, and induce an immune response against ROR-1 expressing cancer cells. In some embodiments, the chimeric antigen receptors described herein are expressed on the surface of natural killer cells (NK cells or genetically modified or engineered NK cells). In certain embodiments, the NK cells are CD56 positive cells. CD56 positivity can be determined, for example, by flow cytometry analysis of a cell population and is defined as at least 10-fold, 100-fold, or 1,000-fold more cells compared to cells stained with an isotype control antibody. In certain specific embodiments, the NK cells are primary NK cells. In certain specific embodiments, the NK cells include NK cell lines. In certain embodiments, the NK cells are autologous to an individual being treated with a CAR expressing the NK cells of the present disclosure. In certain embodiments, the NK cells are allogeneic to an individual being treated with a CAR expressing the NK cells of the present disclosure. In certain embodiments, the NK cells are syngeneic to an individual being treated with a CAR expressing the NK cells of the present disclosure. NK cells can be derived from any suitable source of bone marrow, induced pluripotent stem cells, peripheral blood mononuclear cells, fetal or placental cells.

[0117] Also provided herein is a recombinant nucleic acid encoding a recombinant protein or chimeric antigen receptor provided herein, including embodiments thereof. In some embodiments, the chimeric antigen receptor is encoded by a single recombinant nucleic acid that forms part of an expression vector. Any suitable expression vector capable of being transfected and expressed in immune cells (e.g., CD8+ T cells, CD4+ T cells, CD56+ immune cells, NK cells, or genetically modified or engineered NK cells) is contemplated. Exemplary and / or preferred expression vectors can include viral expression vectors (e.g., expression vectors of adenovirus, adeno-associated virus, alphavirus, herpes virus, lentivirus, etc.). In some embodiments, the adenovirus is a replication-deficient and non-immunogenic virus, which is typically achieved by targeted deletions of selected viral proteins (e.g., E1, E2b, E3 proteins). For example, the recombinant nucleic acid can be placed in a non-viral vector (e.g., a mammalian expression vector) and transfected into T cells using any commonly used transfection method. In other examples, the recombinant nucleic acid is included in a viral vector such that viral particles containing the recombinant nucleic acid infect T cells and deliver the recombinant nucleic acid to the T cells. In still other examples, when the recombinant nucleic acid is a self-replicating RNA-based vector, the self-replicating RNA-based vector can be formulated with a pharmaceutically acceptable carrier (e.g., in a buffer or cell culture medium, preferably containing an RNase inhibitor) such that the self-replicating RNA-based vector can be delivered to cells in a naked form by direct contact of the self-replicating RNA-based vector with the cell membrane. In some embodiments, the self-replicating RNA-based vector can be conjugated to a carrier molecule. Exemplary carrier molecules include protein A, protein G, protein Z, albumin, refolding albumin, nanoparticles (e.g., quantum dots, gold nanoparticles, magnetic nanoparticles, nanotubes, polymeric nanoparticles, dendrimers, etc.), or beads (e.g., polystyrene beads, latex beads, Dynabeads, etc.). Preferably, the nanoparticles and / or beads have dimensions of less than 1 μm, preferably less than 100 nm.In other embodiments, the self-replicating RNA-based vector can bind to microparticles (e.g., PLG RG503 (50:50 molar ratio of lactide / glycolide)), where the self-replicating RNA-based vector can be absorbed into the microparticles for further delivery to cells. In still other embodiments, the self-replicating RNA-based vector can be encapsulated within liposomes (e.g., PEG-based liposomes, etc.) to protect the self-replicating RNA-based vector from RNase digestion, and the RNA-based vector can be delivered by fusing the liposomes to the target cell membrane.

[0118] In embodiments, the recombinant nucleic acid encoding the recombinant protein or chimeric antigen receptor provided herein can be inserted into a vector having a cassette for gene editing (e.g., a CRISPR-CAS expression vector). Another exemplary expression vector can include a transposon-based expression system (e.g., the Sleeping Beauty system disclosed in Deninger et al., PLOS ONE, June 1, 2015).

[0119] Pharmaceutical composition The immune cells expressing the chimeric antigen receptor and / or the recombinant nucleic acid encoding the chimeric antigen receptor can optionally be further formulated as a pharmaceutical composition together with any pharmaceutically acceptable carrier (e.g., as a sterile injectable composition for immune cells expressing the chimeric antigen receptor, a pharmaceutically acceptable salt for the recombinant nucleic acid encoding the chimeric antigen receptor, etc.). The dosage or cell titer of the pharmaceutical composition can vary depending on the treatment protocol, treatment regimen, treatment conditions, etc., but an example of the dosage or cell titer of the pharmaceutical composition is at least 1×10 3 cells / ml, preferably at least 1×10 5 cells / ml, more preferably at least 1×10 6 cells / ml, and can include a cell titer of at least 1 ml, preferably at least 5 ml, more preferably and at least 20 ml per administration unit.

[0120] In some embodiments, the pharmaceutical composition may comprise homogeneous cells or a plurality thereof (e.g., CD8+ T cells expressing a chimeric antigen receptor, CD4+ T cells expressing a chimeric antigen receptor, NK cells expressing a chimeric antigen receptor, etc.). In other embodiments, the composition is a mixture of heterogeneous cells (e.g., a mixture in a ratio of 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1, etc., such as CD8+ T cells expressing a chimeric antigen receptor and NK cells expressing a chimeric antigen receptor). In this pharmaceutical composition, the ratio of different types of cells can vary based on the type of cancer, the patient's age, gender, or health status, the size of the tumor, and the number of the patient's cells. The actual amount effective for a particular use will vary, inter alia, depending on the condition being treated. When administered in a method for treating a disease, the recombinant proteins described herein contain an amount of the active ingredient effective to achieve a desired result, e.g., modulation of the activity of a target molecule and / or reduction, elimination, or delay in the progression of disease symptoms. Determination of a therapeutically effective amount of the compounds of the present invention is well within the ability of those skilled in the art, particularly in light of the detailed disclosure herein.

[0121] The dosage and frequency (single or multiple administrations) administered to a mammal can vary depending on various factors, such as whether the mammal is suffering from another disease, as well as its route of administration, the size, age, gender, health, weight, body mass index, and diet of the recipient, the nature and extent of the symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of co-treatment, complications with the disease being treated, or other health-related problems. Other treatment regimens or agents may be used in conjunction with the methods and compounds of the present invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.

[0122] For any compound provided herein (e.g., recombinant protein, nucleic acid), a therapeutically effective amount can first be determined from cell culture assays. The target concentration, when measured using the methods described herein or methods known in the art, will be the concentration of the active compound capable of achieving the methods described herein. As is well known in the art, an effective amount for use in humans can also be determined from animal models. For example, a human dosage can be formulated to achieve a concentration found to be effective in animals. The dosage in humans can be adjusted as described above by monitoring effectiveness and adjusting the dosage up or down. Based on the above methods and other methods, adjusting the dosage to achieve maximum effectiveness in humans is well within the ability of one of ordinary skill in the art.

[0123] Dosages may vary depending upon the requirements of the patient and the compound being used. In the context of the present invention, the dosage administered to a patient should be sufficient to provide a beneficial therapeutic response in the patient over time. The size of the dosage will also be determined by the presence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the ability of one of ordinary skill in the art. Generally, treatment is initiated at a lower dosage less than the optimum dosage of the compound. Thereafter, the dosage is increased in small increments until the optimum effect under the circumstances is reached.

[0124] Dosage and interval can be adjusted individually to provide the levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen appropriate to the severity of the individual's disease state.

[0125] Using the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that causes substantially no toxicity and is also effective in treating the clinical symptoms presented by a particular patient. This plan should preferably involve a careful selection of the active compound by considering factors such as the potency of the compound, relative bioavailability, the weight of the patient, and the presence and severity of any adverse side effects.

[0126] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that can be included in the compositions of the present invention that assist in the administration of the active agent to a subject and its absorption by the subject, without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, standard saline, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline (such as Ringer's solution), alcohol, oil, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and, if desired, can be mixed with auxiliaries such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for affecting osmotic pressure, buffering agents, coloring agents, and / or aromatic substances that do not react detrimentally with the compounds of the present invention. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0127] The term "pharmaceutically acceptable salts" is derived from various organic and inorganic counterions well known in the art and refers to salts such as, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functional group, refers to salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc.

[0128] The term "formulation" is intended to include the combination of an active compound with a capsule encapsulation material as a carrier for providing the capsule, and the active ingredient, whether or not containing other carriers in the capsule, is surrounded by and thus associated with the carrier. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0129] The pharmaceutical formulation is preferably in unit dosage form. In such form, the formulation is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a packaged formulation, and the package contains individual amounts of the formulation such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or troche itself, or can be a form in which any of these are packaged in an appropriate number. The unit dosage form can be that of a frozen dispersion.

[0130] Treatment method The CAR-T cell composition described herein is useful for treating cancer or tumors in an individual. Accordingly, described herein is a method of treating cancer in a subject in need thereof. This method includes administering to the subject a therapeutically effective amount of CAR-T cells provided herein, including its embodiments, thereby treating the subject's cancer or tumor. The CAR T cell composition is generally administered with one or more pharmaceutically acceptable excipients, carriers, or diluents.

[0131] In certain embodiments, disclosed herein are anti-ROR-1 CAR T cells and anti-ROR-1 CAR-T cell compositions useful for the treatment of cancer or tumors. Treatment refers to a method of attempting to improve or ameliorate a condition being treated. With respect to cancer, treatment includes, but is not limited to, a decrease in tumor volume, a decrease in the growth of tumor volume, an increase in progression-free survival, or an increase in overall mean life expectancy. In certain embodiments, treatment will result in remission of the cancer being treated. In certain embodiments, treatment includes use as a prophylactic or maintenance dose for the purpose of preventing recurrence or progression of a previously treated cancer or tumor. Those skilled in the art understand that these individuals are considered to have been treated, although not all individuals respond equally or adequately to the treatment administered.

[0132] In certain embodiments, the cancer or tumor treated using the anti-ROR-1 CAR-T cells described herein is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the blood cancer is leukemia or lymphoma. In certain embodiments, the leukemia or lymphoma includes B-cell leukemia, lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), multiple myeloma, B-cell lymphoblastic acute lymphoblastic leukemia (B-ALL), mantle cell lymphoma (MCL), T-cell lymphoblastic acute lymphoblastic leukemia T-ALL, and combinations thereof. In certain embodiments, the solid cancer / tumor includes ovarian cancer, colon cancer, lung cancer, breast cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, adrenal cancer, and combinations thereof. In certain embodiments, the cancer is recurrent or resistant to at least one other treatment.

[0133] In certain embodiments, the anti-ROR-1 CAR T cells and anti-ROR-1 CAR-T cell compositions can be administered to a subject in need thereof by any route suitable for administration of a cell-containing pharmaceutical composition, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracranially. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally.

[0134] The anti-ROR-1 CAR T cells and anti-ROR-1 CAR-T cell compositions can be administered according to a suitable dosing schedule. In certain embodiments, the CAR T cells are administered once, and subsequent administrations are dependent on clinical criteria. If an individual does not respond or only responds partially, the patient can be administered the anti-ROR-1 CAR-T cell composition a second, third, or fourth time until the desired clinical response is observed. The dosage of CAR-T cells generally includes at least 1×10 6 cells, but 5×10 8would contain fewer than [number] cells. The cells can be administered based on the total viable PBMC of an individual transduced with the CAR construct. In certain embodiments, a single dose can contain from 1 million transduced PBMC to 100 million transduced PBMC. In certain embodiments, a single dosage can be 1 million transduced PBMC to 2 million transduced PBMC, 1 million transduced PBMC to 3 million transduced PBMC, 1 million transduced PBMC to 4 million transduced PBMC, 1 million transduced PBMC to 5 million transduced PBMC, 1 million transduced PBMC to 6 million transduced PBMC, 1 million transduced PBMC to 7 million transduced PBMC, 1 million transduced PBMC to 8 million transduced PBMC, 1 million transduced PBMC to 9 million transduced PBMC, 1 million transduced PBMC to 10 million transduced PBMC, 1 million transduced PBMC to 50 million transduced PBMC, 1 million transduced PBMC to 100 million transduced PBMC, 2 million transduced PBMC to 3 million transduced PBMC, 2 million transduced PBMC to 4 million transduced PBMC, 2 million transduced PBMC to 5 million transduced PBMC, 2 million transduced PBMC to 6 million transduced PBMC, 2 million transduced PBMC to 7 million transduced PBMC, 2 million transduced PBMC to 8 million transduced PBMC, 2 million transduced PBMC to 9 million transduced PBMC, 2 million transduced PBMC to 10 million transduced PBMC, 2 million transduced PBMC to 50 million transduced PBMC, 2 million transduced PBMC to 100 million transduced PBMC, 3 million transduced PBMC to 4 million transduced PBMC, 3 million transduced PBMC to 5 million transduced PBMC,3 million transduced PBMCs to 6 million transduced PBMCs, 3 million transduced PBMCs to 7 million transduced PBMCs, 3 million transduced PBMCs to 8 million transduced PBMCs, 3 million transduced PBMCs to 9 million transduced PBMCs, 3 million transduced PBMCs to 10 million transduced PBMCs, 3 million transduced PBMCs to 50 million transduced PBMCs, 3 million transduced PBMCs to 100 million transduced PBMCs, 4 million transduced PBMCs to 5 million transduced PBMCs, 4 million transduced PBMCs to 6 million transduced PBMCs, 4 million transduced PBMCs to 7 million transduced PBMCs, 4 million transduced PBMCs to 8 million transduced PBMCs, 4 million transduced PBMCs to 9 million transduced PBMCs, 4 million transduced PBMCs to 10 million transduced PBMCs, 4 million transduced PBMCs to 50 million transduced PBMCs, 4 million transduced PBMCs to 100 million transduced PBMCs, 5 million transduced PBMCs to 6 million transduced PBMCs, 5 million transduced PBMCs to 7 million transduced PBMCs, 5 million transduced PBMCs to 8 million transduced PBMCs, 5 million transduced PBMCs to 9 million transduced PBMCs, 5 million transduced PBMCs to 10 million transduced PBMCs, 5 million transduced PBMCs to 50 million transduced PBMCs, 5 million transduced PBMCs to 100 million transduced PBMCs, 6 million transduced PBMCs to 7 million transduced PBMCs, 6 million transduced PBMCs to 8 million transduced PBMCs, 6 million transduced PBMCs to 9 million transduced PBMCs, 6 million transduced PBMCs to 10 million transduced PBMCs, 6 million transduced PBMCs to 50 million transduced PBMCs6 million transduced PBMCs to 100 million transduced PBMCs, 7 million transduced PBMCs to 8 million transduced PBMCs, 7 million transduced PBMCs to 9 million transduced PBMCs, 7 million transduced PBMCs to 10 million transduced PBMCs, 7 million transduced PBMCs to 50 million transduced PBMCs, 7 million transduced PBMCs to 100 million transduced PBMCs, 8 million transduced PBMCs to 9 million transduced PBMCs, 8 million transduced PBMCs to 10 million transduced PBMCs, 8 million transduced PBMCs to 50 million transduced PBMCs, 8 million transduced PBMCs to 100 million transduced PBMCs, 9 million transduced PBMCs to 10 million transduced PBMCs, 9 million transduced PBMCs to 50 million transduced PBMCs, 9 million transduced PBMCs to 100 million transduced PBMCs, 10 million transduced PBMCs to 50 million transduced PBMCs, 10 million transduced PBMCs to 100 million transduced PBMCs, or 50 million transduced PBMCs to 100 million transduced PBMCs. In certain embodiments, the single dose comprises 1 million transduced PBMCs, 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs, 9 million transduced PBMCs, 10 million transduced PBMCs, 50 million transduced PBMCs, or 100 million transduced PBMCs. In certain embodiments, the single dose is at least 1 million transduced PBMCs, 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs,It includes 9 million transduced PBMCs, 10 million transduced PBMCs, or 50 million transduced PBMCs. In certain embodiments, the single dose can be up to 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs, 9 million transduced PBMCs, 10 million transduced PBMCs, 50 million transduced PBMCs, or 100 million transduced PBMCs.,

[0135] Case by case, more or less cells can be used according to the transduction efficiency of individual T cells. In certain embodiments, the single dose comprises from 1 million CAR-T cells to 100 million CAR-T cells. In certain embodiments, the single dose is 1 million CAR-T cells to 2 million CAR-T cells, 1 million CAR-T cells to 3 million CAR-T cells, 1 million CAR-T cells to 4 million CAR-T cells, 1 million CAR-T cells to 5 million CAR-T cells, 1 million CAR-T cells to 6 million CAR-T cells, 1 million CAR-T cells to 7 million CAR-T cells, 1 million CAR-T cells to 8 million CAR-T cells, 1 million CAR-T cells to 9 million CAR-T cells, 1 million CAR-T cells to 10 million CAR-T cells, 1 million CAR-T cells to 50 million CAR-T cells, 1 million CAR-T cells to 100 million CAR-T cells, 2 million CAR-T cells to 3 million CAR-T cells, 2 million CAR-T cells to 4 million CAR-T cells, 2 million CAR-T cells to 5 million CAR-T cells, 2 million CAR-T cells to 6 million CAR-T cells, 2 million CAR-T cells to 7 million CAR-T cells, 2 million CAR-T cells to 8 million CAR-T cells, 2 million CAR-T cells to 9 million CAR-T cells, 2 million CAR-T cells to 10 million CAR-T cells, 2 million CAR-T cells to 50 million CAR-T cells, 2 million CAR-T cells to 100 million CAR-T cells, 3 million CAR-T cells to 4 million CAR-T cells, 3 million CAR-T cells to 5 million CAR-T cells, 3 million CAR-T cells to 6 million CAR-T cells, 3 million CAR-T cells to 7 million CAR-T cells, 3 million CAR-T cells to 8 million CAR-T cells, 3 million CAR-T cells to 9 million CAR-T cells, 3 million CAR-T cells to 10 million CAR-T cells, 3 million CAR-T cells to 50 million CAR-T cells, 3 million CAR-T cells to 100 million CAR-T cells, 4 million CAR-T cells to 5 million CAR-T cells,4 million to 6 million CAR-T cells, 4 million to 7 million CAR-T cells, 4 million to 8 million CAR-T cells, 4 million to 9 million CAR-T cells, 4 million to 10 million CAR-T cells, 4 million to 50 million CAR-T cells, 4 million to 100 million CAR-T cells, 5 million to 6 million CAR-T cells, 5 million to 7 million CAR-T cells, 5 million to 8 million CAR-T cells, 5 million to 9 million CAR-T cells, 5 million to 10 million CAR-T cells, 5 million to 50 million CAR-T cells, 5 million to 100 million CAR-T cells, 6 million to 7 million CAR-T cells, 6 million to 8 million CAR-T cells, 6 million to 9 million CAR-T cells, 6 million to 10 million CAR-T cells, 6 million to 50 million CAR-T cells, 6 million to 100 million CAR-T cells, 7 million to 8 million CAR-T cells, 7 million to 9 million CAR-T cells, 7 million to 10 million CAR-T cells, 7 million to 50 million CAR-T cells, 7 million to 100 million CAR-T cells, 8 million to 9 million CAR-T cells, 8 million to 10 million CAR-T cells, 8 million to 50 million CAR-T cells, 8 million to 100 million CAR-T cells, 9 million to 10 million CAR-T cells, 9 million to 50 million CAR-T cells, 9 million to 100 million CAR-T cells, 10 million to 50 million CAR-T cells, 10 million to 100 million CAR-T cells, or 50 million to 100 million CAR-T cells. In certain embodiments,The single-dose administration contains 1 million CAR-T cells, 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, 50 million CAR-T cells, or 100 million CAR-T cells. In certain embodiments, the single-dose administration contains at least 1 million CAR-T cells, 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, or 50 million CAR-T cells. In certain embodiments, the single-dose administration contains at most 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, 50 million CAR-T cells, or 100 million CAR-T cells.,

[0136] In certain embodiments, the population of anti-ROR-1 CAR-T cells of the present disclosure is included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the CAR-T cells of the present disclosure are administered by suspension in a sterile isotonic solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris), a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188, a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40, an amino acid, such as glycine or arginine, an antioxidant, such as ascorbic acid, methionine, or a chelating agent, such as EDTA or EGTA. The CAR-T cells can be formulated and buffered at a specific pH, generally about 7.0 to about 8.0. In certain embodiments, the cells are buffered at a physiological pH of about 7.4 (averaging about 7.35 to 7.45).

[0137] Also described herein is a method of treating an individual who has developed one or more serious adverse events associated with CAR T cell therapy, comprising administering a bolus injection of siltuximab. Such administration can prevent the CAR T cells from interacting with their targets and attenuate their activity.

[0138] Also described herein is a kit comprising one or more of the nucleic acids encoding the CAR-T cells described herein in a suitable container and one additional component selected from: instructions for use, diluent, excipient, carrier, and device for administration).

[0139] In certain embodiments, described herein is a method of preparing a cancer therapeutic agent comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the disclosure. In certain embodiments, described herein is a method of preparing a cancer therapeutic agent for storage or transport comprising lyophilizing one or more antibodies of the disclosure.

Examples

[0140] The following exemplary examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0141] Example 1: Chimeric Antigen Receptor-Modified T Cells (CAR-T) Targeting ROR-1 A series of monoclonal antibodies targeting the ROR-1 antigen have been previously described, and this target is the subject of ongoing clinical trials in patients with CLL, MCL, and breast cancer using the anti-human ROR-1 mAb UC-961 (siltuximab) in combination with standard chemotherapy. The CARs constructed and tested in these examples utilize the complementarity-determining regions (CDRs) and variable regions (V H / V L ) of the siltuximab antibody. The siltuximab antibody and the sequences encoding its CDRs and VH / VL are disclosed in WO2014 / 031174.

[0142] There are several unmet needs in the CAR-T space for the treatment of cancer. The CAR T cells described herein address these unmet needs.

[0143] Treatment failure The recurrence rate of patients after CAR-T therapy is increasing, and in many cases, it is due to mutations or loss of the target tumor antigen (e.g., CD19), allowing cancer cells to escape CAR-T cells. Since the expression of ROR-1 is associated with an aggressive tumor phenotype, using ROR-1 as a target may reduce antigen-negative recurrence, and mutations or loss of the antigen may reduce the aggressiveness of cancer cells and make them more susceptible to chemotherapy.

[0144] Safety concerns Persistent CAR-T safety issues include deaths that may be related to activation of normal cells expressing the target antigen ("on-target / off-tumor"). Previous studies have shown that siltuximab did not bind to normal human tissue in GLP tissue cross-reactivity tests, and there are no severe adverse events associated with siltuximab - reported only in clinical trials.

[0145] An additional means of therapeutic intervention is to use the antigen-binding domains of these anti-human ROR-1 antibodies, including UC-961, as the target portion of the CAR. Figure 1A shows a conceptual diagram of anti-ROR-1 CAR-T cells interacting with ROR-1-expressing cancer cells (CLL). Figure 1B shows a schematic diagram of different non-limiting embodiments of the CAR of the present disclosure. The CAR of the present disclosure describes the light chain CDR of anti-human ROR-1 mAb 4A5 or UC-961 bound to the heavy chain CDR of the same mAb by a specific linker that generates a high-affinity single-chain (scFv) molecule that specifically binds to human ROR-1 with sufficient affinity to activate intracellular signaling and cytotoxicity of transduced T lymphocytes. A series of protein spacers made from IgG4 were generated to bind the scFv to the transmembrane domain. These spacers allow the anti-ROR-1 scFv binding domain to have sufficient flexibility to optimally bind to the target ROR-1 antigen.

[0146] Still referring to FIG. 1B, the extracellular antigen-binding domain from the CAR binds to the CD28 transmembrane domain. This transmembrane domain is then linked to intracellular activation domains derived from CD28 and / or CD137, which are used either alone (second-generation CAR) or in combination (third-generation CAR). These activation domains are then linked to the T cell receptor activation domain contained within the CD3 zeta chain (CD3ζ chain). Thus, the anti-ROR-1 CAR (shown in FIGS. 1A, 1B, 1C) is, in order from the N-terminus, a leader that directs the construct to the cell surface, a light chain CDR of either 4A5 or UC-961 mAb, a linker that connects the light and heavy chains, a heavy chain CDR of 4A5 or UC-961 mAb that is complemented by the corresponding light chain molecule, a defined-length spacer generated from IgG4 that includes the IgG4 hinge region, the CD28 transmembrane region, intracellular activation domains of CD28 and 41-BB (CD137) that extend to the carboxy terminus of the molecule, either individually or tandemly, and are expressed as a single polypeptide containing the T cell ζ chain. FIGS. 2A and 2B show vector schematic diagrams depicting CARs with different spacer domains.

[0147] As shown in Figure 3, the CAR described in this specification can be transposon-based or contained in a lentiviral vector and introduced into target lymphocytes using standard transfection or transduction techniques. To maximize stable transduction and long-term expression of the CAR product, retroviral delivery systems such as murine gamma and human lentiviruses are used. Currently, the third-generation lentiviral 4-plasmid system (Addgene, Inc.) is used to transgenically engineer ROR-1 T lymphocytes that express the CAR. This third-generation system is based on the lentiviral vector system described in Naldini et al, "Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector" Proc Natl Acad Sci USA. 1996 Oct 15;93(21):11382-11388. This 4-plasmid system includes plasmid 1 - gag / pol, plasmid 2 - rev, plasmid 3 VSV-G protein, and plasmid 4, and the transfer plasmid contains a nucleic acid sequence encoding an anti-ROR1 CAR inserted using a restriction site in the polylinker.

[0148] Figures 4A and 4B show the steps for generating anti-ROR-1 CAR-expressing T cells. Referring to Figure 4A, T cells are isolated from peripheral blood mononuclear cells (PBMCs), followed by activation with CD3 / Cd28 nanoparticles, transduction with lentivirus, and optionally, an analysis step to verify CAR expression and specificity.

[0149] Figure 5A shows the flow cytometry analysis of whole blood PBMC. Figure 5B shows the purified CD8+ and CD4+ populations. Figure 6A shows that activation by CD3 / CD28 results in activation of both CD4+ (80%) and CD8+ (63%) cells, and Figure 6B shows that lentiviral transduction results in dose-dependent transduction efficiency of 10% (MOI 1) or 28% (MOI 3) of CD8+ T cells, or 15% (MOI 1) or 34% (MOI 3) of CD4+ T cells. Figures 7A and 7B show the time course of anti-ROR-1 CAR expression after lentiviral transduction of T lymphocytes.

[0150] Example 2: In vitro activity of chimeric antigen receptor-modified T cells (CAR-T) targeting ROR-1 Several chimeric antigen receptors (CARs) targeting the cancer-related antigen ROR-1 were created according to the above scheme and used to transduce cytotoxic T lymphocytes.

[0151] To generate multiple candidates for anti-human ROR1 T cell CARs, second-generation and third CAR constructs with different signaling domains and spacer domains were generated. To test these constructs, candidate CAR-Ts were generated and grown in serum-free growth medium supplemented with IL-2. Using this protocol, CAR-T cells from more than 20 healthy donors were generated and their activities against lymphoid cancers were investigated in vitro using chromium release and impedance assays. Second-generation CARs using the 4-1BB costimulatory domain had activity even at low effector-to-target (E:T) ratios and eliminated both primary B cell leukemia and lymphoma, as well as tumor cell lines expressing the ROR1 target.

[0152] These T cell CARs demonstrated specific activity targeting ROR-1, which is expressed in both hematological and solid tumor cancers. As shown in Figure 8A, T lymphocytes expressing anti-ROR-1 CAR were effective in killing leukemic MEC-1 / ROR1 cells at different effector-to-target (E:T) ratios. These results were generated using a 4-hour chromium release assay. In this test, the anti-ROR1 CAR expressing a hinge and CH3 spacer demonstrated the greatest activity. For Figure 8A, "activation only" (leftmost bar, ET9) are T cells isolated from PBMC or blood products and have undergone the same activation and cell culture processes as CAR-T cells, but without lentiviral CAR transduction. "Long UC961" (second bar from the left) refers to a construct utilizing a CH2CH3 spacer that includes both spacer #3 (SEQ ID NO: 29) and the CH2 and CH3 regions of hIgG4. "CH3" (third bar from the left) refers to a CAR-T construct utilizing a spacer CH3 that includes spacer #3 and the CH3 region of human IgG4. "Hinge" (fourth bar from the left) refers to a CAR-T construct utilizing spacer #3 (hinge) without the human IgG4 CH2 and CH3 domains. Figure 8B shows a 4-hour chromium release assay using non-ROR-1 expressing MEC-1 cells. This data indicates that the ROR-1 CAR is specific and kills only based on the presence of ROR-1 on the cell surface of target cells. The bars in Figure 8B are ordered as in Figure 8A.

[0153] Figure 8C shows the effect on the cytotoxicity of second or third generation anti-ROR-1 CARs. Figure 8C shows an experimental setup similar to Figures 8A and 8B, except that the hinge or CH3 is compared as a second generation CAR (4-1BB intracellular signaling domain only) or third generation CAR (both 4-1BB and CD28 intracellular signaling domains). There is no difference between the second and third generation of constructs with a full CH3-hinge, but the second generation format 第 3 世代The format showed improved killing compared to the second-generation format of constructs having only hinges and no CH3. Figure 8D shows no killing of MEC-1 cells that do not express ROR-1, indicating that these CARs are similarly specific. Figure 8E. Cell killing of MEC1-ROR1 cells by CAR T from two different donors.

[0154] As shown in Figure 9A, the anti-ROR-1 CAR kills MCF-7 ROR-1 cells. This figure shows specific killing of MCF-7 / ROR-1 breast cancer cells by anti-ROR-1 T cell CARs with different spacer lengths generated as described in Example 1. These results were generated through the use of a 96-hour impedance assay (ACEA). In this test, anti-ROR-1 CARs expressing the hinge domain and CH3 domain showed the greatest activity against ROR-1 positive cells. When used in Figure 9A, "CH3" refers to a CAR-T construct utilizing spacer #3 (SEQ ID NO: 29) and a spacer CH3 containing the CH3 region of human IgG4, "hinge" refers to a CAR-T construct utilizing spacer #3 (hinge) without human IgG4 CH2 or CH3, and "ET1" refers to the 1:1 effector-to-target ratio of anti-ROR-1 CAR-T cells to target cells in a cytotoxicity assay. Figure 9B shows that the killing observed in Figure 9A is specific since MCF-7 cells that do not express ROR-1 are not killed by anti-ROR-1 CAR-T cells. Figure 9C shows killing from CAR T cells generated from two different healthy donors.

[0155] Figure 10 shows that T lymphocytes expressing anti-ROR-1 CAR kill MEC1 / ROR-1 cells and Jeko cells that naturally express ROR-1 at different effector-to-target ratios.

[0156] Example 3: In Vivo Activity of Chimeric Antigen Receptor-Modified T Cells (CAR-T) Targeting ROR-1 To test the ROR1 T cell CAR in vivo, a luciferase / RFP dual-specific lentiviral expression vector was created to modify human MEC1-ROR1 leukemia cells (Yu et al., J Clin Invest, 2015). When injected into immunodeficient NSG mice, MEC1-ROR1 cells infiltrate the bone marrow (femur), kidney, spleen, and liver, resulting in progressive and fatal leukemia / lymphoma. As shown in Figure 11A, when injected into immunodeficient mice, this highly active cell line rapidly proliferates in the bone marrow (femur), kidney, spleen, liver, and blood, and the animals die of leukemia within approximately 3 weeks. The in vivo experiment was conducted as shown in Figure 11B (top), and briefly, mice were injected with 1×10 6 ROR-1 / MEC1-luciferase cells on day 1, followed by 1×10 6 or 3×10 6 anti-ROR-1 CAR-T cells on day 4. Tumor growth was measured using the bioluminescence of the modified MEC cells at subsequent time points.

[0157] Mice injected with tumor cells responded in a dose-dependent manner to the second-generation CAR-T cells from in vitro tests. 3×10 6 anti-ROR-1 CAR T cells were able to eliminate ROR-1 / MEC cells expressing luciferase, as shown in Figures 12 and 13. By week 3, rapidly proliferating leukemia cells were eliminated from the bone marrow, and the remaining leukemia cells could not be detected by week 4, and the animals survived for more than 90 days compared to approximately 21 days in the absence of these CAR-T cells. Figure 14 shows that anti-ROR1 CAR-T cells were the major components of the animals' bone marrow and kidney by days 11 and 18. Furthermore, the concentration of CAR-T cells was substantially higher in mice pretreated with MEC-1 ROR1 cells (left panel) than in the mock control (right panel), indicating an increase in the proliferation of CAR-T in animals with leukemia cells.

[0158] Summarizing the results shown here, it is as follows.

[0159] Without a pre-treatment regimen or cytokine supplementation regimen, 3×10 unloaded 6 Anti-ROR1 4-1BB CAR T cells were injected once, expanded in animals with MEC1-ROR1 xenografts, and returned to the site of MEC1-ROR1 disease activity.

[0160] By week 4, rapidly proliferating leukemia cells were removed from major tissue reservoirs including bone marrow, kidney, and spleen.

[0161] Animals treated with CAR-T survived for more than 90 days, compared to approximately 21 days in animals administered only non-transduced activated T cells or in animals that were untreated controls.

[0162] The administered CAR T product was highly active and detected in mouse tissues several months after injection.

[0163] These tests have been extended to other lymphoid tumor cell lines including JeKo cells expressing ROR-1.

[0164] Although preferred embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision many variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments described herein may be employed in the practice of the present invention.

[0165] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated herein by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference. Definitions contained in the text incorporated by reference are excluded to the extent they are inconsistent with the definitions of the present disclosure.

[0166] Unofficial Sequence Listing UC961 VL: DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK (SEQ ID NO: 21) Linker #3: GGGGSGSTSGSGKPGSGEGSTKGGGGGS (SEQ ID NO: 24) UC961 VH: QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS (SEQ ID NO: 27) Spacer #3 (or "hinge / short"): VDESKYGPPCPPCP (SEQ ID NO: 29) Spacer CH3: VDESKYGPPCPPCPLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41) Spacer CH2CH3 (or "long / full"): VDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 42) UC961 CDR L1: KSISKY (SEQ ID NO: 43) UC961 CDR L2: SGS (SEQ ID NO: 44) UC961 CDR L3: QQHDESPY (SEQ ID NO: 45) UC961 CDR H1: GYAFTAYN (SEQ ID NO: 46) UC961 CDR H2: FDPYDGGS (SEQ ID NO: 47) UC961 CDR H3: GWYYFDY (SEQ ID NO: 48) 4A5 CDR L1: PDINSY (SEQ ID NO: 49) 4A5 CDR L2: RAN (SEQ ID NO: 50) 4A5 CDR L3: LQYDEFPYT (SEQ ID NO: 51) 4A5 CDR H1: GFTFSSYA (SEQ ID NO: 52) 4A5 CDR H2: ISRGGTT (SEQ ID NO: 53) 4A5 CDR H3: YDYDGYYAMDY (SEQ ID NO: 54) Full-length human ROR-1 protein (SEQ ID NO: 55): MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWMEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL 21 - amino - acid stretch of human ROR - 1 containing 138 - glutamic acid (SEQ ID NO: 56): VATNGKEVVSSTGVLFVKFGP 15 - amino - acid stretch of human ROR - 1 containing 138 - glutamic acid (SEQ ID NO: 57): EVVSSTGVLFVKFGP

[0167] P embodiment P embodiment 1. (i) (a) A light - chain variable domain comprising CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and (b) a heavy - chain variable domain comprising CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, and (ii) a transmembrane domain, a recombinant protein.

[0168] P embodiment 2. (i) (a) A light - chain variable domain comprising CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and (b) a heavy - chain variable domain comprising CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54, and (ii) a transmembrane domain, a recombinant protein.

[0169] P embodiment 3. The recombinant protein according to P embodiment 1, wherein the C - terminus of the light - chain variable domain is bound to the N - terminus of the heavy - chain variable domain.

[0170] P embodiment 4. The recombinant protein according to any one of P embodiments 1 to 3, wherein the light - chain variable domain is covalently bound to the heavy - chain variable domain via a chemical linker.

[0171] P embodiment 5. The recombinant protein according to any one of P embodiments 1 to 4, wherein the chemical linker is a peptide linker.

[0172] P Embodiment 6. The recombinant protein according to P Embodiment 5, wherein the peptide linker has the sequence of SEQ ID NO: 24.

[0173] P Embodiment 7. The recombinant protein according to any one of P Embodiments 1 to 6, wherein the C-terminus of the heavy chain variable domain is bound to the N-terminus of the transmembrane domain.

[0174] P Embodiment 8. The recombinant protein according to any one of P Embodiments 1 to 7, wherein the heavy chain variable domain is covalently bound to the transmembrane domain via a spacer domain.

[0175] P Embodiment 9. The recombinant protein according to P Embodiment 8, wherein the spacer domain contains an antibody domain.

[0176] P Embodiment 10. The recombinant protein according to P Embodiment 9, wherein the antibody domain contains a hinge domain, a constant heavy chain 3 (CH3) domain, a constant heavy chain 2 (CH2) domain, or any combination thereof.

[0177] P Embodiment 11. The recombinant protein according to P Embodiment 9 or 10, wherein the antibody domain consists of a hinge domain.

[0178] P Embodiment 12. The recombinant protein according to P Embodiment 9 or 10, wherein the antibody domain consists of a hinge domain and a constant heavy chain 3 (CH3) domain.

[0179] P Embodiment 13. The recombinant protein according to P Embodiment 9 or 10, wherein the antibody domain consists of a hinge domain, a constant heavy chain 3 (CH3) domain, and a constant heavy chain 2 (CH2) domain.

[0180] P Embodiment 14. The recombinant protein according to any one of P Embodiments 9 to 13, wherein the spacer domain contains the sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0181] P Embodiment 15. The recombinant protein according to any one of P Embodiments 9 to 13, wherein the spacer domain has the sequence of SEQ ID NO: 29, SEQ ID NO: 41 or SEQ ID NO: 42.

[0182] P Embodiment 16. The recombinant protein according to any one of P Embodiments 1 to 15, wherein the light chain variable domain contains the sequence of SEQ ID NO: 21.

[0183] P Embodiment 17. The recombinant protein according to any one of P Embodiments 1 to 16, wherein the light chain variable domain has the sequence of SEQ ID NO: 21.

[0184] P Embodiment 18. The recombinant protein according to any one of P Embodiments 1 to 17, wherein the heavy chain variable domain contains the sequence of SEQ ID NO: 27.

[0185] P Embodiment 19. The recombinant protein according to any one of P Embodiments 1 to 18, wherein the heavy chain variable domain contains the sequence of SEQ ID NO: 27.

[0186] P Embodiment 20. The recombinant protein according to any one of P Embodiments 2 to 13, wherein the light chain variable domain contains the sequence of SEQ ID NO: 19.

[0187] P Embodiment 21. The recombinant protein according to any one of P Embodiments 2 to 13 or 20, wherein the light chain variable domain has the sequence of SEQ ID NO: 19.

[0188] P Embodiment 22. The recombinant protein according to any one of P Embodiments 2 to 13, wherein the light chain variable domain contains the sequence of SEQ ID NO: 20.

[0189] P Embodiment 23. The recombinant protein according to any one of P Embodiments 2 to 13 or 22, wherein the light chain variable domain has the sequence of SEQ ID NO: 20.

[0190] P Embodiment 24. The recombinant protein according to any one of P Embodiments 2 to 13, wherein the heavy chain variable domain contains the sequence of SEQ ID NO: 25.

[0191] P Embodiment 25. The recombinant protein according to any one of P Embodiments 2 to 13 or 24, wherein the heavy chain variable domain has the sequence of SEQ ID NO: 25.

[0192] P Embodiment 26. The recombinant protein according to any one of P Embodiments 2 to 13, wherein the heavy chain variable domain contains the sequence of SEQ ID NO: 26.

[0193] P Embodiment 27. The recombinant protein according to any one of P Embodiments 2 to 13 or 26, wherein the heavy chain variable domain has the sequence of SEQ ID NO: 26.

[0194] P Embodiment 28. The recombinant protein according to any one of P Embodiments 1 to 27, wherein the transmembrane domain is a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, or a CD3ζ transmembrane domain, or any combination thereof.

[0195] P Embodiment 29. The recombinant protein according to any one of P Embodiments 1 to 28, wherein the transmembrane domain is a CD28 transmembrane domain.

[0196] P Embodiment 30. The recombinant protein according to P Embodiment 29, wherein the CD28 transmembrane domain contains the sequence of SEQ ID NO: 32.

[0197] P Embodiment 31. The recombinant protein according to P Embodiment 29, wherein the CD28 transmembrane domain has the sequence of SEQ ID NO: 32.

[0198] P Embodiment 32. The recombinant protein according to any one of P Embodiments 1 to 31, further comprising one or more intracellular co-stimulatory signaling domains.

[0199] P Embodiment 33. The one or more intracellular costimulatory signaling domains are the 4-1BB intracellular costimulatory signaling domain, the CD28 intracellular costimulatory signaling domain, the ICOS intracellular costimulatory signaling domain, the OX-40 intracellular costimulatory signaling domain, or any combination thereof, and the recombinant protein according to P Embodiment 32.

[0200] P Embodiment 34. The recombinant protein according to P Embodiment 32 or 33, which contains the 4-1BB intracellular costimulatory signaling domain.

[0201] P Embodiment 35. The recombinant protein according to P Embodiment 34, wherein the 4-1BB intracellular costimulatory signaling domain contains the sequence of SEQ ID NO: 33.

[0202] P Embodiment 36. The recombinant protein according to P Embodiment 34 or 35, wherein the 4-1BB intracellular costimulatory signaling domain has the sequence of SEQ ID NO: 33.

[0203] P Embodiment 37. The recombinant protein according to P Embodiment 32, which contains the CD28 intracellular costimulatory signaling domain and the 4-1BB intracellular costimulatory signaling domain.

[0204] P Embodiment 38. The recombinant protein according to any one of P Embodiments 1 to 37, which further contains an intracellular T cell signaling domain.

[0205] P Embodiment 39. The recombinant protein according to P Embodiment 38, wherein the intracellular T cell signaling domain is the CD3ζ intracellular T cell signaling domain.

[0206] P Embodiment 40. The recombinant protein according to P Embodiment 39, wherein the CD3ζ intracellular T cell signaling domain contains the sequence of SEQ ID NO: 34.

[0207] P Embodiment 41. The recombinant protein according to P Embodiment 39, wherein the CD3ζ intracellular T cell signaling domain has the sequence of SEQ ID NO: 34.

[0208] P Embodiment 42. The recombinant protein according to any one of P Embodiments 1 to 41, wherein the recombinant protein binds to cells.

[0209] P Embodiment 43. The recombinant protein according to P Embodiment 42, wherein the cell is a cancer cell.

[0210] P Embodiment 44. The recombinant protein according to P Embodiment 43, wherein the cancer cell is a leukemia cell, a lymphoma cell, a chronic lymphocytic leukemia (CLL) cell, an adult acute myeloid leukemia (AML) cell, an acute lymphoblastic leukemia (ALL) cell, a mantle cell lymphoma (MCL) cell, a multiple myeloma cell, an ovarian cancer cell, a colon cancer cell, a lung cancer cell, a skin cancer cell, a pancreatic cancer cell, a testicular cancer cell, a bladder cancer cell, a uterine cancer cell, a prostate cancer cell, a breast cancer cell, or an adrenal cancer cell.

[0211] P Embodiment 45. The recombinant protein according to any one of P Embodiments 1 to 44, wherein the recombinant protein forms part of a cell.

[0212] P Embodiment 46. The recombinant protein according to any one of P Embodiments 1 to 46, wherein the recombinant protein forms part of a cell.

[0213] P Embodiment 47. An isolated nucleic acid encoding the recombinant protein according to any one of P Embodiments 1 to 46.

[0214] P Embodiment 48. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant protein according to any one of P Embodiments 1 to 46 and a pharmaceutically acceptable excipient.

[0215] P Embodiment 49. A method for treating cancer in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the recombinant protein according to one of P Embodiments 1 to 46, thereby treating the cancer of the subject.

[0216] Embodiment Embodiment 1. A chimeric antigen receptor, an antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1, and the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, or the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54, an antigen-binding region, a spacer domain, wherein the spacer domain comprises a spacer having a length of 10 to 240 amino acids, a spacer domain, a transmembrane domain, and an intracellular domain, a chimeric antigen receptor comprising.

[0217] Embodiment 2. The chimeric antigen receptor according to Embodiment 1, wherein the spacer has a length of 14 to 120 amino acids.

[0218] Embodiment 3. The chimeric antigen receptor according to Embodiment 1 or 2, wherein the light chain variable domain is at the N-terminus of the heavy chain variable domain.

[0219] Embodiment 4. The chimeric antigen receptor according to Embodiment 3, wherein the light chain variable domain is covalently bound to the heavy chain variable domain via a polypeptide linker.

[0220] Embodiment 5. The chimeric antigen receptor according to Embodiment 4, wherein the polypeptide linker consists of the sequence of SEQ ID NO: 24.

[0221] Embodiment 6. The chimeric antigen receptor according to any one of Embodiments 1 to 5, wherein the spacer domain contains an antibody domain.

[0222] Embodiment 7. The chimeric antigen receptor according to Embodiment 6, wherein the antibody domain contains an immunoglobulin hinge domain, an immunoglobulin constant heavy chain 3 (CH3) domain, an immunoglobulin constant heavy chain 2 (CH2) domain, or any combination thereof.

[0223] Embodiment 8. The chimeric antigen receptor according to Embodiment 7, wherein the antibody domain consists of the immunoglobulin hinge domain.

[0224] Embodiment 9. The chimeric antigen receptor according to Embodiment 7, wherein the antibody domain consists of the immunoglobulin hinge domain and the immunoglobulin constant heavy chain 3 (CH3) domain.

[0225] Embodiment 10. The chimeric antigen receptor according to Embodiment 7, wherein the antibody domain consists of the immunoglobulin hinge domain, the immunoglobulin constant heavy chain 3 (CH3) domain, and the immunoglobulin constant heavy chain 2 (CH2) domain.

[0226] Embodiment 11. The chimeric antigen receptor according to Embodiment 7, wherein the spacer domain contains the sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0227] Embodiment 12. The recombinant protein according to Embodiment 7, wherein the spacer domain consists of the sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0228] Embodiment 13. The chimeric antigen receptor according to any one of Embodiments 7 to 12, wherein the light chain variable domain contains an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21.

[0229] Embodiment 14. The chimeric antigen receptor according to any one of Embodiments 7 to 12, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 21.

[0230] Embodiment 15. The chimeric antigen receptor according to any one of Embodiments 7 to 12, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 27.

[0231] Embodiment 16. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 27.

[0232] Embodiment 17. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 19.

[0233] Embodiment 18. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 19.

[0234] Embodiment 19. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 20.

[0235] Embodiment 20. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 20.

[0236] Embodiment 21. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 25.

[0237] Embodiment 22. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 25.

[0238] Embodiment 23. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26.

[0239] Embodiment 24. The chimeric antigen receptor according to any one of Embodiments 1 to 12, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 26.

[0240] Embodiment 25. The chimeric antigen receptor according to any one of Embodiments 1 to 24, wherein the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof.

[0241] Embodiment 26. The chimeric antigen receptor according to Embodiment 25, wherein the transmembrane domain is a CD28 transmembrane domain.

[0242] Embodiment 27. The chimeric antigen receptor according to Embodiment 25, wherein the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 32.

[0243] Embodiment 28. The chimeric antigen receptor according to Embodiment 25, wherein the CD28 transmembrane domain consists of the sequence of SEQ ID NO: 32.

[0244] Embodiment 29. The chimeric antigen receptor according to any one of Embodiments 1 to 28, wherein the intracellular domain comprises one or more intracellular co-stimulatory signaling domains, one or more intracellular T cell signaling domains, or one or more intracellular co-stimulatory signaling domains and one or more intracellular T cell signaling domains.

[0245] Embodiment 30. The chimeric antigen receptor according to embodiment 29, wherein the one or more intracellular co-stimulatory signaling domains are 4-1BB intracellular co-stimulatory signaling domain, CD28 intracellular co-stimulatory signaling domain, ICOS intracellular co-stimulatory signaling domain, OX-40 intracellular co-stimulatory signaling domain, or any combination thereof.

[0246] Embodiment 31. The chimeric antigen receptor according to embodiment 29, comprising a 4-1BB intracellular co-stimulatory signaling domain.

[0247] Embodiment 32. The chimeric antigen receptor according to embodiment 31, wherein the 4-1BB intracellular co-stimulatory signaling domain comprises the sequence of SEQ ID NO: 33.

[0248] Embodiment 33. The chimeric antigen receptor according to embodiment 31, wherein the 4-1BB intracellular co-stimulatory signaling domain consists of the sequence of SEQ ID NO: 33.

[0249] Embodiment 34. The chimeric antigen receptor according to embodiment 29, comprising a CD28 intracellular co-stimulatory signaling domain and a 4-1BB intracellular co-stimulatory signaling domain.

[0250] Embodiment 35. The chimeric antigen receptor according to any one of embodiments 1 to 34, further comprising an intracellular T cell signaling domain.

[0251] mbodiment 36. The chimeric antigen receptor according to embodiment 35, wherein the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0252] Embodiment 37. The chimeric antigen receptor according to embodiment 36, wherein the CD3ζ intracellular T cell signaling domain comprises the sequence of SEQ ID NO: 34.

[0253] Embodiment 38. The chimeric antigen receptor according to embodiment 36, wherein the CD3ζ intracellular T cell signaling domain consists of the sequence of SEQ ID NO: 34.

[0254] Embodiment 39. The chimeric antigen receptor according to any one of Embodiments 1 to 38, which binds to cells expressing ROR-1.

[0255] Embodiment 40. A nucleic acid encoding the chimeric antigen receptor according to any one of Embodiments 1 to 39.

[0256] Embodiment 41. The nucleic acid according to Embodiment 40, wherein the nucleic acid is a viral vector.

[0257] Embodiment 42. The nucleic acid according to Embodiment 41, wherein the nucleic acid vector is a lentiviral vector.

[0258] Embodiment 43. A cell comprising the nucleic acid according to any one of Embodiments 40 to 42.

[0259] Embodiment 44. A cell expressing the chimeric antigen receptor according to any one of Embodiments 1 to 39.

[0260] Embodiment 45. The cell according to Embodiment 43 or 44, wherein the cell is a T lymphocyte.

[0261] Embodiment 46. The cell according to Embodiment 45, wherein the T lymphocyte is a CD8-positive T lymphocyte.

[0262] Embodiment 47. The cell according to Embodiment 43 or 44, wherein the cell is a natural killer cell.

[0263] Embodiment 48. A pharmaceutical composition comprising a therapeutically effective amount of the cell population according to any one of Embodiments 43 to 47 and a pharmaceutically acceptable diluent, carrier, or excipient.

[0264] Embodiment 49. The pharmaceutical composition according to Embodiment 48, formulated for intravenous injection.

[0265] Embodiment 50. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual the pharmaceutical composition according to Embodiment 48 or 49.

[0266] Embodiment 51. The method according to Embodiment 50, wherein the cancer comprises leukemia, lymphoma, chronic lymphocytic leukemia (CLL), adult acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer.

[0267] Embodiment 52. The method according to Embodiment 50 or 51, wherein the method further comprises administering siltuximab to the individual.

[0268] Embodiment 53. The method according to Embodiment 52, wherein the siltuximab and the pharmaceutical composition according to Embodiment 48 or 49 are administered separately.

[0269] Embodiment 54. The pharmaceutical composition according to Embodiment 48 or 49 for use in a method for treating cancer in an individual.

[0270] Embodiment 55. The pharmaceutical composition for use according to Embodiment 54, wherein the cancer comprises leukemia, lymphoma, chronic lymphocytic leukemia (CLL), adult acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer.

[0271] Embodiment 56. A chimeric antigen receptor, An antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1, and the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain, The light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, or the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54, an antigen-binding region, a transmembrane domain, an intracellular domain, the intracellular domain comprising an intracellular T cell signaling domain and an intracellular co-stimulatory domain selected from 4-1BB, ICOS, OX-40, and combinations thereof, an intracellular domain, a chimeric alternative receptor.

[0272] Embodiment 57. The chimeric antigen receptor according to embodiment 56, comprising a spacer domain, the spacer domain comprising a spacer having a length of 10 to 200 amino acids.

[0273] Embodiment 58. The chimeric antigen receptor according to embodiment 57, wherein the spacer has a length of 14 to 120 amino acids.

[0274] Embodiment 59. The chimeric antigen receptor according to embodiment 56 or 58, wherein the light chain variable domain is at the N-terminus of the heavy chain variable domain.

[0275] Embodiment 60. The chimeric antigen receptor according to embodiment 59, wherein the light chain variable domain is covalently bound to the heavy chain variable domain via a polypeptide linker.

[0276] Embodiment 61. The chimeric antigen receptor according to embodiment 60, wherein the polypeptide linker consists of the sequence of SEQ ID NO: 24.

[0277] Embodiment 62. The chimeric antigen receptor according to any one of Embodiments 56 to 61, wherein the spacer domain comprises an antibody domain.

[0278] Embodiment 63. The chimeric antigen receptor according to Embodiment 62, wherein the antibody domain comprises an immunoglobulin hinge domain, an immunoglobulin constant heavy chain 3 (CH3) domain, an immunoglobulin constant heavy chain 2 (CH2) domain, or any combination thereof.

[0279] Embodiment 64. The chimeric antigen receptor according to Embodiment 63, wherein the antibody domain consists of the immunoglobulin hinge domain.

[0280] Embodiment 65. The chimeric antigen receptor according to Embodiment 63, wherein the antibody domain consists of an immunoglobulin hinge domain and the immunoglobulin constant heavy chain 3 (CH3) domain.

[0281] Embodiment 66. The chimeric antigen receptor according to Embodiment 63, wherein the antibody domain consists of the immunoglobulin hinge domain, the immunoglobulin constant heavy chain 3 (CH3) domain, and the immunoglobulin constant heavy chain 2 (CH2) domain.

[0282] Embodiment 67. The chimeric antigen receptor according to Embodiment 63, wherein the spacer domain comprises the sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0283] Embodiment 68. The recombinant protein according to Embodiment 63, wherein the spacer domain consists of the sequence of SEQ ID NO: 29, SEQ ID NO: 41, or SEQ ID NO: 42.

[0284] Embodiment 69. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21.

[0285] Embodiment 70. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 21.

[0286] Embodiment 71. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 27.

[0287] Embodiment 72. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 27.

[0288] Embodiment 73. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 19.

[0289] Embodiment 74. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 19.

[0290] Embodiment 75. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 20.

[0291] Embodiment 76. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the light chain variable domain consists of the sequence of SEQ ID NO: 20.

[0292] Embodiment 77. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 25.

[0293] Embodiment 78. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 25.

[0294] Embodiment 79. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26.

[0295] Embodiment 80. The chimeric antigen receptor according to any one of Embodiments 56 to 68, wherein the heavy chain variable domain consists of the sequence of SEQ ID NO: 26.

[0296] Embodiment 81. The chimeric antigen receptor according to any one of Embodiments 56 to 80, wherein the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof.

[0297] Embodiment 82. The chimeric antigen receptor according to Embodiment 81, wherein the transmembrane domain is a CD28 transmembrane domain.

[0298] Embodiment 83. The chimeric antigen receptor according to Embodiment 82, wherein the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 32.

[0299] Embodiment 84. The chimeric antigen receptor according to Embodiment 82, wherein the CD28 transmembrane domain consists of the sequence of SEQ ID NO: 32.

[0300] Embodiment 85. The chimeric antigen receptor according to any one of Embodiments 56 to 84, wherein the intracellular domain comprises a 4-1BB intracellular co-stimulatory signaling domain.

[0301] Embodiment 86. The chimeric antigen receptor according to Embodiment 85, wherein the 4-1BB intracellular co-stimulatory signaling domain comprises the sequence of SEQ ID NO: 33.

[0302] Embodiment 87. The chimeric antigen receptor according to Embodiment 85, wherein the 4-1BB intracellular costimulatory signaling domain consists of the sequence of SEQ ID NO: 33.

[0303] Embodiment 88. The chimeric antigen receptor according to any one of Embodiments 56 to 87, comprising a CD28 intracellular costimulatory signaling domain and a 4-1BB intracellular costimulatory signaling domain.

[0304] Embodiment 89. The chimeric antigen receptor according to any one of Embodiments 56 to 88, wherein the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0305] Embodiment 90. The chimeric antigen receptor according to Embodiment 89, wherein the CD3ζ intracellular T cell signaling domain comprises the sequence of SEQ ID NO: 34.

[0306] Embodiment 91. The chimeric antigen receptor according to Embodiment 89, wherein the CD3ζ intracellular T cell signaling domain consists of the sequence of SEQ ID NO: 34.

[0307] Embodiment 92. The chimeric antigen receptor according to any one of Embodiments 56 to 91, wherein the chimeric antigen receptor binds to cells expressing ROR-1.

[0308] Embodiment 93. A nucleic acid encoding the chimeric antigen receptor according to any one of Embodiments 56 to 92.

[0309] Embodiment 94. The nucleic acid according to Embodiment 93, wherein the nucleic acid is a viral vector.

[0310] Embodiment 95. The nucleic acid according to Embodiment 94, wherein the nucleic acid vector is a lentiviral vector.

[0311] Embodiment 96. A cell comprising the nucleic acid according to any one of Embodiments 93 to 95.

[0312] Cell expressing the chimeric antigen receptor according to any one of Embodiments 56 to 92.

[0313] Embodiment 98. The cell according to Embodiment 96 or 97, wherein the cell is a T lymphocyte.

[0314] Embodiment 99. The cell according to Embodiment 98, wherein the T lymphocyte is a CD8-positive T lymphocyte.

[0315] Embodiment 100. The cell according to Embodiment 96 or 97, wherein the cell is a natural killer cell.

[0316] Embodiment 101. A pharmaceutical composition comprising a therapeutically effective amount of the cell population according to any one of Embodiments 96 to 100, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0317] Embodiment 102. The pharmaceutical composition according to Embodiment 101, formulated for intravenous injection.

[0318] Embodiment 103. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual the pharmaceutical composition according to Embodiment 101.

[0319] Embodiment 104. The method according to Embodiment 103, wherein the cancer includes leukemia, lymphoma, chronic lymphocytic leukemia (CLL), adult acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer.

[0320] Embodiment 105. The method according to Embodiment 103 or 104, further comprising administering siltuximab to the individual.

[0321] Embodiment 106. The method according to Embodiment 105, wherein the siltuximab and the pharmaceutical composition according to Embodiment 101 or 102 are administered separately.

[0322] The pharmaceutical composition according to Embodiment 101 or 102 for use in a method of treating cancer in an individual.

[0323] Embodiment 108. The pharmaceutical composition for use according to Embodiment 107, wherein the cancer includes leukemia, lymphoma, chronic lymphocytic leukemia (CLL), adult acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer.

[0324] SEQ ID NOs: 44 and 50, which are the sequences skipped when converting from the st.25 compliant sequence listing to the st.26 compliant sequence listing, are shown below. <210> 44 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 44 Ser Gly Ser 1 <210> 50 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 50 Arg Ala Asn 1

Claims

**Claim 1**: A chimeric antigen receptor, comprising: i. An antigen-binding region, wherein the antigen-binding region specifically binds to ROR-1, and the antigen-binding region comprises a light chain variable domain and a heavy chain variable domain, (a) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 43, CDR L2 set forth in SEQ ID NO: 44, and CDR L3 set forth in SEQ ID NO: 45, the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 46, CDR H2 set forth in SEQ ID NO: 47, and CDR H3 set forth in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 set forth in SEQ ID NO: 49, CDR L2 set forth in SEQ ID NO: 50, and CDR L3 set forth in SEQ ID NO: 51, the heavy chain variable domain comprises CDR H1 set forth in SEQ ID NO: 52, CDR H2 set forth in SEQ ID NO: 53, and CDR H3 set forth in SEQ ID NO: 54, an antigen-binding region; ii. A spacer domain comprising SEQ ID NO: 29 or SEQ ID NO: 41; iii. A transmembrane domain; iv. An intracellular domain ; and the chimeric antigen receptor binds to cells expressing ROR-1. **Claim 2**: The chimeric antigen receptor according to claim 1, wherein the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, SEQ ID NO: 19, or SEQ ID NO:

20. **Claim 3**: The chimeric antigen receptor according to claim 1, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27, SEQ ID NO: 25, or SEQ ID NO:

26. **Claim 4**: The chimeric antigen receptor according to claim 1, wherein the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, the amino acid sequence of SEQ ID NO: 32, or any combination thereof. **Claim 5**: The chimeric antigen receptor according to claim 1, wherein the intracellular domain comprises an intracellular co-stimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof. **Claim 6**: The intracellular costimulatory signaling domain is the 4-1BB intracellular costimulatory signaling domain, the CD28 intracellular costimulatory signaling domain, the ICOS intracellular costimulatory signaling domain, the OX-40 intracellular costimulatory signaling domain, the amino acid sequence of SEQ ID NO: 33, the CD28 intracellular costimulatory signaling domain, the 4-IBB intracellular costimulatory signaling domain, or any combination thereof. The chimeric antigen receptor according to claim 5. **Claim 7**: The intracellular costimulatory signaling domain further comprises an intracellular T cell signaling domain, an intracellular T cell signaling domain which is a CD3ζ intracellular T cell signaling domain, the amino acid sequence of SEQ ID NO: 34, or any combination thereof. The chimeric antigen receptor according to claim 5 or 6. **Claim 8**: A nucleic acid encoding the chimeric antigen receptor according to claim 1. **Claim 9**: Optionally, the nucleic acid is a viral vector. Optionally, the viral vector is a lentiviral vector. The nucleic acid according to claim 8. **Claim 10**: A cell expressing the chimeric antigen receptor according to claim 1. **Claim 11**: The cell is a T lymphocyte. The cell according to claim 10. **Claim 12**: The T lymphocyte is a CD4+ T lymphocyte or a CD8+ T lymphocyte. The cell according to claim 11. **Claim 13**: The cell is a natural killer cell. The cell according to claim 10. **Claim 14**: A pharmaceutical composition comprising a therapeutically effective amount of the cell according to any one of claims 10 to 13, and a pharmaceutically acceptable diluent, carrier, or excipient. **Claim 15**: The pharmaceutical composition according to claim 14 for use in the treatment of cancer. **Claim 16**: The cancer includes leukemia, lymphoma, chronic lymphocytic leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, mantle cell lymphoma, ovarian cancer, colon cancer, lung cancer, skin cancer, pancreatic cancer, testicular cancer, bladder cancer, uterine cancer, prostate cancer, or adrenal cancer. The pharmaceutical composition for use in the treatment of cancer according to claim 15. **Claim 17**: The cancer is a CD19-negative cancer or has reduced CD19 expression as a result of previous treatment targeting CD19. The pharmaceutical composition for use in the treatment of cancer according to claim 15. A pharmaceutical composition for use in the treatment of cancer according to claim 15, which is administered to an individual previously treated with a therapeutic agent targeting CD19.

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